Medicinal components comprising human parathyroid hormone and medicinal compositions for nasal administration containing the components

ABSTRACT

A medical component comprising a human parathyroid hormone peptide or its derivative, and acetic acid contained at a concentration less than its chemical equivalent with respect to the human parathyroid hormone peptide or to its derivative. Since in the medical component acetic acid, which is present as a salt of or attached to the peptide or its derivative, has been reduced to an amount less than chemical equivalent with respect to the human parathyroid hormone peptide or its derivative, a medical component, which is highly stable and will ensure an excellent use feeling when introduced into a pharmaceutical composition, is obtained.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical component based onhuman parathyroid hormone which has an excellent stability, and ensuresa good use feeling when used as a component of a pharmaceuticalcomposition. In a further aspect, this invention relates to apharmaceutical composition based on human parathyroid hormone forintranasal administration which is suitable for a long term use.

BACKGROUND ART

The peptides of human parathyroid hormone (to be referred to as “hPTH”hereinafter) are biologically active peptides which are responsible forbone metabolism, and have an strong activity to develop bones (Aurbachet al., Recent Progr. Horm. Res., 1972, vol. 28, p. 35). The hPTH is apeptide typically composed of 84 amino acid residues (hPTH(1-84)). Aderivative, hPTH(1-34), from hPTH(1-84) composed of 34 amino acidresidues designated as amino acid Nos. 1-34 of hPTH(1-84) has been alsoknown to have the same pharmacological activity as does hPTH(1-84)(Tregear et al., Hoppe-Seyler's Z. Physiol. Chem., 1974, vol. 355, p.415). The amino acid sequences of hPTH(1-84) and hPTH(1-34) are given asSequence Nos. 1 and 2 in the Sequence Listing, respectively.

Calcitonin and bisphosphonate or therapeutic agents used for thetreatment of osteoporosis exhibit their therapeutic effects byinhibiting bone resorption, whereas hPTH(1-84) and hPTH(1-34) stimulatebone formation, or bone metabolism involved in the bone formation. Thus,those peptides have been expected to serve as a new therapeutic agent ofosteoporosis (Lane et al., J. Clin. Invest., 1998, vol. 102, pp.1627-1633).

It has been reported, when hPTH(1-34) is subcutaneously applied tohumans at a certain dose once weekly, it will increase the bone mineralcontent, while, when the same agent is similarly applied at a dose onefifth the previous one but once daily for five days successively, itwill also increase the bone of mineral density (BMD) significantly (Soneet al., Miner Electrolyte Metab., 1995, vol. 21, pp. 232-235). In animalexperiments it has been shown that hPTH, when given subcutaneously at acertain dose once weekly, will cause less BMP to bones than is observedwhen it is given divisional (Tawaragi et al., OsteoporosisInternational, vol. 6, suppl. 1, 1996, p. 245). This suggests a maximumtherapeutic effect from hPTH will be obtained when hPTH is given dailyat a small dose over a long period than when it is given at a large dosefor a short period with a long interval between successive doses. If asmall dose of hPTH given continuously over a long period will ensure antherapeutic effect equal to or more excellent than does a large dosegiven intermittently for a short period, this prescription will be alsodesirable from the view-point of small doses of hPTH being probably freefrom the adverse effects on the digestive and cardiovascular organswhich are known for high dose administrations of hPTH.

Also, injections would be unsuitable to be used for the treatment of thepatient with osteoporosis who will usually require a long termtreatment, because the patient should then receive the treatment underthe management of a physician; feel a more or less pain duringtreatment; and visit the physician's office regularly for treatmentwhich would be a burden to the patient.

In view of this, there is a need for a nasal drug which would allow thepatient to easily take it at home daily over a long period with no unduepain and burden imposed upon the patient.

However, for a nasal drug to be safely used continuously over a longperiod it is absolutely necessary that the drug should be smoothlyabsorbed through the nasal mucosa; have no irritating action against thenasal mucosa; and give an excellent use feeling, because the nasalmucosa is very sensitive to the irritating action of a medicine or anadditive thereof. Particularly, for such a drug as the one prepared fromhPTH which is expected to give a therapeutic effect when used over along period, an excellent use feeling is very important when it isintended to be used for intranasal administration. In order to prepare anasal drug which is acceptable even when used continuously over a longperiod, it is important to select the active agent in the form of amedicament or of its salt, and proper additives, to determine theireffective concentrations, and to optimize their combination. The factorsresponsible for the use feeling of a nasal drug involve the odor andirritating activity of the drug. Thus, the kinds of medicaments oradditives used for a nasal drug and their concentrations are verylimited.

There is a commercially available product prepared from hPTH, that is,an injection containing a 5-acetate of hPTH(1-34) which is used as adiagnostic agent for assaying the functional activity of parathyroid(whose generic name is teriparatide acetate and which is provided byAsahi Kasei Kogyo Corp.). However, no intranasal drug based on hPTH isavailable that will give a satisfactory use feeling in terms of odor andirritability.

Japanese Patent Laid-Open No. 64-16799 describes that, when hPTH(1-34)is purified, it is mixed with acetic acid which is used for thepurification process, and the acetic acid content in individual productsvary widely from lot to lot, making it difficult to obtain productscontaining a uniform amount of acetic acid, and that introduction ofacetic acid to the product will lead to a reduced activity of theproduct.

The same document further discloses a method suitable for improving thestability of hPTH(1-34) wherein a lyophilized composition of hPTH(1-34)based on the use of tartaric acid is utilized. However, tartaric acid isso highly acidic that an agent containing it will not be suitable forintranasal administration.

Japanese Patent Laid-Open No. 2-111 discloses a powdery composition forintranasal administration based on a water soluble organic acid whichhas been developed to improve the nasal absorption of hPTH(1-34) abiologically active peptide such as hPTH (1-34). However, thecomposition would not be suitable for a long term use, because it willdirectly irritate the nasal mucosa, depending on the kind and amount ofcoexistent organic acid.

As discussed above, no intranasal drug based on hPTH that is acceptableeven when used over a long period, and ensures an excellent use feelingas well as good stability and absorption has been developed.

DISCLOSURE OF INVENTION

This invention aims at providing a pharmaceutical component based onhPTH which is highly stable, when used as a component of apharmaceutical composition, and gives an excellent use feeling. In astill other aspect, this invention aims at providing a pharmaceuticalcomposition for intranasal administration based on hPTH which will allowa long term use.

To meet the object of providing a pharmaceutical composition forintranasal administration as described above, the present inventors hadstudied hard, and reached a finding that an hPTH preparationconventionally prepared is unsatisfactory because it will give an acidicodor and irritation when administered to the nasal mucosa, and that thisinconvenience is ascribed to acetic acid which is used in thepurification process and exists in very small amount as the constituentof a salt of hPTH or an adherent. Based on this finding, they prepared apharmaceutical component whose acetic acid content is reduced ascompared with the previous one, assessed it and surprisingly found thatthe component in question is highly stable, gives an excellent usefeeling when incorporated in a pharamceutical composition for, andpresents with a property of being safely combined with the appropriateamounts of functional components which will be added for the improvementof absorption and stability, as well as with a carrier and excipientwhich are usually used during preparing medicines. Thus, they achievedthis invention.

To put it more specifically, the present invention relates to apharmaceutical component comprising hPTH and acetic acid whose contentis kept less than a certain chemical equivalent with respect to theweight of hPTH. In a still other aspect, this invention relates to apharamceutical composition for intranasal administration which containshPTH as its active ingredient, and which also contains acetic acid whosecontent is kept less than a certain chemical equivalent with respect tothe weight of hPTH.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reverse phase HPLC chromatogram obtained from anhPTH(1-34) preparation as acetic acid content being 9.5% after it hasbeen stored at 40° C. for six months.

FIG. 2 shows a reverse phase HPLC chromatogram obtained from anhPTH(1-34) preparation as acetic acid content being 2.9% after it hasbeen stored at 40° C. for six months.

FIG. 3 shows the amounts of decomposition products (by-products) B, Cand D after hPTH(1-34) preparations contained various acetic contentsacid have been stored at 80° C. for 15 hours.

FIG. 4 shows the purity levels of hPTH(1-34) of the hPTH(1-34)preparations contained various acetic acid content after thepreparations have been stored at 80° C. for 15 hours.

FIG. 5 shows a reverse phase HPLC chromatogram of an hPTH(1-84)preparation as acetic acid content being 12.3% after it has been storedat 80° C. for 15 hours.

FIG. 6 shows the amounts of decomposition products (by-products) afterhPTH(1-84) preparations contained various acetic acid contents have beenstored at 80° C. for 15 hours.

FIG. 7 shows the purity levels of hPTH(1-84) of the hPTH(1-84)preparations containing various acetic acid content after thepreparations have been stored at 80° C. for 15 hours.

DESCRIPTION OF THE EMBODIMENTS

According to this invention, hPTH includes peptides which are involvedin bone metabolism, have a strong stimulating effect on the formation ofbones, and have an activity to increase the concentration of calcium inserum, that is, a natural type hPTH(1-84) comprising 84 amino acidresidues, and its derivatives.

For example, the hPTH may include hPTH(1-84) (Biochemistry 17,5723(1978); Kimura et al., Biochem. Biophys. Res. Commun., vol. 114, p.493, 1983), hPTH(1-38) (Japanese Patent Laid-Open No. 57-81448),hPTH(1-34) (Japanese Patent Laid-Open No. 9-29600; Takai et al., PeptideChemistry, p. 187, 1979), hPTH(1-34)NH₂ (Japanese Patent Laid-Open No.58-96052), [Nle^(8,18)]hPTH(1-34) and [Nle^(8,18), Tyr³⁴]hPTH(1-34)(Japanese Patent Laid-Open No. 55-113753), [Nle8.18]hPTH(1-34)NH₂(Japanese Patent Laid-Open No. 61-24598), [Nle^(8,18),Tyr³⁴]hPTH(1-34)NH₂ (Japanese Patent Laid-Open No. 60-34996), hPTH(1-37)(Japanese Patent Presentation [Kohyo] No. 5-505594), hPTH(2-84),hPTH(3-84), hPTH(4-84), hPTH(5-84), hPTH(6-84), hPTH(7-84), andhPTH(8-84) (Japanese Patent Presentation [Kohyo] No. 4-505259), etc.These hPTHs can be obtained by the methods based on genetic engineeringtechniques or chemical synthesis techniques as described in the abovedocuments, or by the methods equivalent with the former.

Generally speaking, when a physiologically active peptide is purifiedbased on the genetic engineering technique or chemical synthesistechnique, column chromatography is used. However, because hPHT is abasic peptide, it would be adsorbed to resin constituting a column if itwere unscrupulously applied to the column. To prevent the adsorption ofhPTH and raise its solubility, an acid is used as an eluent. For apeptide to serve as a material of a pharmaceutical composition, thepeptide must be incorporated in a lyophilized composition which willserve as a starting material for preparation of the pharmaceuticalcomposition. To meet this requirement, the acid must be volatile, whichlimits the range of usable acids.

For illustration, let's assume, for example, hydrochloric acid is usedfor the present purpose. It is highly acidic even at a lowconcentration, readily causes accessory reactions such as hydrolysis,and is highly corrosive. Thus, hydrochloric acid is not suitable for thepresent purpose. If an organic acid is employed for the present purpose,it may be selected from trifluoroacetic acid, formic acid and aceticacid that have a low boiling point. However, it is undesirable toincorporate trifluoriacetic acid in a pharmaceutical composition interms of safety. Formic acid is also limited in its use because of itsreducing activity, and is not compatible with a peptide such as hPTH.

By contrast, acetic acid is the most appropriate to serve as a materialfor a pharmaceutical composition, on account of its safety and chemicalproperties, and has been used as an acid indispensable for the finalstage purification of a basic peptide. For example, the purificationprocess based on reverse phase high-performance liquid columnchromatography (reverse phase HPLC) or on size exclusion columnchromatography has been performed using an eluate containing acetic acidwith a favorable result. During the process, acetic acid is added at aconcentration sufficient to prevent hPTH from being adsorbed to thecolumn, and thus a sample eluated from an aqueous solution of aceticacid, a target peptide fraction, and a lyophilized composition preparedtherefrom contain acetic acid at a concentration above the level atwhich acetic acid exists only as the constituent of a salt of the basicamino acid residues, that is, acetic acid exists not only as theconstituent of hPTH salt, but as an adherent attached.

Namely, acetic acid existing in a pharmaceutical component based on hPTHtakes two forms: it exists as the constituent of a salt of hPTH, and anadherent attached. Since acetic acid is a volatile substance, it isdifficult to keep the content of acetic acid in a lyophilizedcomposition at a constant level, because the content of acetic acid in alyophilized composition varies depending on the lyophilizationcondition, the concentration of acetic acid in the original solutionprior to lyophilization, with respect to the concentration of hPTHcoexistent in the original solution.

The pharmaceutical component of this invention comprises hPTH and aceticacid, in which the content of acetic acid existing as the constituent ofa salt of hPTH and an adherent is reduced. This component, because ofhaving a reduced content of acetic acid, improves the stability of hPTH,and ensures a good use feeling when incorporated in a pharmaceuticalcomposition for intranasal administration, and used as such.

In the pharmaceutical component of this invention, acetic acid with areduced content is defined as acetic acid whose content is reduced belowa certain specified chemical equivalent.

Because hPTH(1-34) contains nine basic amino acid residues (includingtryptophan residue), one molecule of it can bind with nine molecules ofmonovalent acid (acetic acid and others) at maximum to form a salt.However, it also contains four acidic amino acid residues which may bindwith the basic residues to form a salt within the same molecule.Therefore, with regard to hPTH(1-34) of this invention, the remainingfive basic amino acid residues are considered as available for bindingwith acetic acid, from which the expected weight of acetic acid to bindwith one molecule of hPTH(1-34) or a chemical equivalent of acetic acidto hPTH(1-34) is derived. The acetic acid content can be obtained byusing the equation I: the weight of acetic acid ×100 (%)/the weight ofhuman parathyroid hormone peptide, on the weight of acetic acid andhuman parathyroid hormone peptide. The chemical equivalent of aceticacid to hPTH(1-34) is about 7.3% (weight % unless otherwise stated) asthe acetic acid content.

Similarly, since hPTH(1-84) contains 19 basic amino acid residues(including tryptophan) and 12 acidic amino acid residues, in preparing ahPTH(1-84) preparation according to this invention, it is assumed thatthe seven excess basic amino acid residues are available for bindingwith acetic acid in one molecule of hPTH(1-84), which will give theexpected weight of acetic acid to bind with one molecule of hPTH or achemical equivalent of acetic acid to hPTH(1-84). The acetic acidcontent can be obtained by using the equation I, the chemical equivalentof acetic acid to hPTH(1-84) is about 4.5% as the acetic acid content.

Namely, according to this invention, acetic acid contained in an amountless than its chemical equivalent means that of an amount of acetic acidomitted as an adherent, but only as the constituent of a salt of hPTHwhose percent weight is below the chemical equivalent.

The present invention provides a stable pharmaceutical component basedon hPTH by controlling the content of acetic acid thereof, and apharmaceutical composition for intranasal administration containing thepharmaceutical component.

Further, the present invention provides a pharmaceutical component basedon hPTH in which the content of acetic acid is controlled such that itis kept at a specified level, and a medicinal composition for intranasaladministration containing the pharmaceutical component.

Since a peptide is generally unstable in a solution, its lyophilizedproduct is used as a material for a pharmaceutical component. If apeptide such as hPTH which exists as a salt containing acetic acid or avolatile substance as the constituent of the salt, is dissolved in wateror in diluted acetic acid, and the lyophilized product thereof is usedas a material for preparing a pharmaceutical component, the content ofacetic acid in the component will not stay at a constant level, whichposes a problem. This invention enables preparation of an aqueoussolution of hPTH with a reduced content of acetic acid, thereby enablingthe production of an hPTH-based pharmaceutical component consistentlycontaining a specific amount of acetic acid in stable. Thus, thisinvention is advantageous also from the aspect of manufacturingstability.

For a pharmaceutical component according to this invention, the aceticacid content with respect to the weight of hPTH is kept below thechemical equivalent. For example, for the hPTH(1-34)-based componentacetic acid content is kept below about 7.3% with respect to the weightof hPTH(1-34), more preferably about 6.0% or less particularly about4.0% or less from the view-point of stability and utility, or morepreferably about 4.0% or less, particularly about 3.0% or less from theview-point of manufacturing stability. It is not preferable that thecontent of acetic acid were kept at a too low level from the view-pointof manufacturing stability, since the component, although it would givean excellent stability and use feeling, would readily become insolubleat a high pH: hPTH(1-34) has an isoelectric point at 8.2 (pI=8.2). Thecontent of acetic acid is preferably kept at about 0.5% or higher,particularly about 1.0% or higher. On the other hand, forhPTH(1-84)-based component, the acetic acid content is kept below about4.5%, preferably about 3.0% or less from the view-point of stability andutility, more preferably about 0.1% or higher from the view-point ofmanufacturing stability.

The pharmaceutical component of this invention can be produced by anypublicly known method or by any method equivalent with the former.Namely, reduction of the content of acetic acid existing as theconstituent of a salt of hPTH or as an adherent below a specified levelcan be achieved by appropriately introducing a known method such asdialysis, electrodialysis, ion exchange chromatography, size exclusioncolumn chromatography, reverse phase HPLC, etc., into the purificationprocess of hPTH which has been obtained by a genetic engineering-basedtechnique or a chemical synthesis-based technique.

When the excess content of acetic acid is reduced by a method such asdialysis, electrodialysis, ion exchange chromatography, etc., adjustmentof the content of acetic acid to any desired level may be achieved bydirectly monitoring the pH of the hPTH solution, or the concentration ofacetic acid in the solution, so that an hPTH solution containing aceticacid at a desired concentration may be obtained.

For example, adjustment of the acetic acid content in an aqueoussolution of hPTH may be achieved based on the relation of the aceticacid content in the solution to the pH of the solution.

Dialysis may occur as follows: an aqueous solution of hPTH which hasbeen prepared by a genetic engineering-based or chemical synthesis-basedtechnique, or the same aqueous solution whose pH has been adjusted topH5-9, with the addition of an alkaline solution such as an aqueoussolution of sodium hydroxide or ammonia is placed in a dialysis membranein the form of a cylinder which will pass low molecular weightcomponents; the solutes in the solution is subject to dialysis based onsimple diffusion; and the acetic acid content is removed by thisprocess. For example, a solution of hPTH(1-34) (acetic acid contentbeing 2%) is obtained by subjecting a starting solution to dialysisuntil the solution outside the dialysis membrane comes to have a pH ofabout 6.5.

Electrodialysis may occur as follows: an aqueous solution of hPTH whichhas been prepared by a genetic engineering-based or chemicalsynthesis-based technique, or the same aqueous solution whose pH hasbeen adjusted to pH5-9, with the addition of an alkaline solution suchas an aqueous solution of sodium hydroxide or ammonia is allowed tocirculate between two dialysis membranes exposed to an electric fieldwhich will pass components having a molecular weight of 300 or less; andacetic acid ions will migrate to the cathode to accumulate there whilefree hPTH basic ions will migrate to the anode to accumulate there; andacetic acid ions with a low molecular weight are allowed to pass throughthe membranes to the outside, while free hPTH basic ions with a largemolecular weight are allowed to circulate within the dialysis system. Itwill be possible to produce a solution of hPTH containing a desiredconstant amount of acetic acid, by monitoring the pH or ionic strengthof dialysis solution, thereby checking the reduced acetic acid content.For example, an hPTH(1-34) solution (acetic acid content being about 2%)will be obtained by applying electrodialysis to a starting solutionuntil the pH of dialysis solution comes to have a pH of about 6.5.

In ion exchange chromatography, acetic acid is adsorbed by binding to abasic ion exchange resin to be removed. For example, an aqueous solutionof hPTH which has been prepared by a genetic engineering-based orchemical synthesis-based technique, is applied to a basic ion exchangeresin column made of a quaternary or secondary ammonium resin; aceticacid is allowed to be bound to the resin through ion-to-ion binding; anda non-adsorbed fraction simply passing through the column is recoveredto give an hPTH solution with a reduced acetic acid content. It ispossible to obtain an hPTH solution with a specified acetic acid contentby altering the amount of ion exchange resin with respect to the weightof hPTH in the solution. For example, in order to obtain an hPTH(1-34)solution (acetic acid content being about 2%), a resin may be used thathas a weight sufficiently large to alter the pH of eluate to aboutpH6.5.

Size exclusion column chromatography may occur as follows: an aqueoussolution of hPTH which has been prepared by a genetic engineering-basedor chemical synthesis-based technique, or the same aqueous solutionwhose pH has been adjusted to pH5-9, with the addition of an alkalinesolution such as an aqueous solution of sodium hydroxide or ammonia isapplied to a column; an aqueous solution containing an organic solventsuch as acetonitrile is used for eluation; and acetic acid is therebyremoved. It is possible to obtain an hPTH solution with a specifiedacetic acid content by altering the pH of the aqueous solution of hPTHto be applied to a column. For example, to obtain an hPTH(1-34) solution(acetic acid content being about 2%), an aqueous solution of hPTH havingbeen so adjusted as to give a pH of about 6.5, is applied to a column,and a fraction consisting of hPTH(1-34) eluate is recovered.

In reverse phase HPLC, an aqueous solution of hPTH which has beenprepared by a genetic engineering-based or chemical synthesis-basedtechnique, or the same aqueous solution whose pH has been adjusted topH5-9, with the addition of an alkaline solution such as an aqueoussolution of sodium hydroxide or ammonia is served. The solution isapplied to a C18 or C4 column initialized with water; and for examplewater is used as an eluate to elute inorganic salts. Then, an aqueoussolution containing an organic solvent such as acetonitrile is allowedto flow to elute hPTH adsorbed to the column; and an hPTH solution witha reduced acetic acid content is thereby obtained.

It is possible to obtain an hPTH solution with a specified acetic acidcontent by adjusting the pH of an aqueous solution of hPTH to be appliedto a column. It is also possible to obtain an hPTH solution with aspecified acetic acid content by preparing an hPTH solution with a toosmall acetic acid content, for example as low a level as permitted tothe method, and then adding a necessary amount of acetic acid to give anhPTH solution with a specified acetic acid content. For example, anhPTH(1-34) solution from which acetic acid has been excessively removed,is diluted with water to 10 mg/mL; acetic acid is added to the solutionuntil the pH of the solution becomes pH6.5; and an hPTH(1-34) solution(acetic acid content being about 2%) is thereby obtained.

An aqueous solution of hPTH obtained by the method as described above islyophilized by a conventional method to produce a pharmaceuticalcomponent of this invention.

The medicinal component of this invention may include water-solubleorganic acids, or preferably at least one selected from citric acid,adipic acid and glycolic acid, so as to improve the mucosal absorptionof the component. A pharmaceutical component further including such anorganic acid will ensure a high stability, and will also ensure anexcellent use feeling, when administered through a route other thanparenteral routes, or particularly when administered nasally.

Accordingly, the pharmaceutical component of this invention may be usedas a component of a pharmaceutical composition for intranasaladministration suitable for a long term use.

Moreover, the pharmaceutical composition of this invention forintranasal administration has a property of being compatible with widelyvaried functional components as well as with a carrier, excipient,viscosity-increasing agent, preserver, stabilizer, anti-oxidant, binder,disintegrant, humectant, lubricant, colorant, flavoring agent,corrigent, suspendmolding agent, emulsifying agent, solubilizer,buffering agent, tonicity agent, detergent, soothing agent,Sulfur-containing reducing agent etc. Thus, the pharmaceuticalcomposition of this invention well tolerates the addition of variousfunctional components which may be introduced to improve absorption,solid stability, etc., as appropriate.

The carrier or excipient may include substances well or sparinglysoluble to water such as sugars, polysaccharides, dextrins, celluloses,synthesized or semi-synthesized polymers, amino acids, polyamino acids,proteins, and phospholipids.

The sugars (monosaccharides, oligosaccharides) may include, for example,D-mannitol, glucose, lactose, fructose, inositol, sucrose, maltose,etc., while the polysaccharides may include dextran, pullulan, alginicacid, hyaluronic acid, pectic acid, phytic acid, phytin, etc. Thedextrins may include a-cyclodextrin, β-cyclodextrin, γ-cyclodextrin,dextrin, hydroxypropylstarch, hydroxyethylstarch, etc.

The celluloses may include methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.

The synthesized or semi-synthesized polymers may include polyvinylalcohol, carboxyvinyl polymer, polyethylene glycol, polyvinylpyrrolidone (PVP), sodium polyacrylate, polyactic acid, etc.

The amino acids may include glycine, taurine, while the polyamino acidsmay include polyglutamic acid, polyaspartic acid, polyglycine,polyleucine, etc.

The proteins may include gelatin and others. In addition, chitin andchitosan may be included.

Of these carriers or excipients, particularly preferred are sucrose,maltose, α-cyclodextrin, β-cyclodestrin, dextrin, D-mannitol, inositol,lactose, dextran, methylcellulose, hydroxypropylcellulose, polyvinylalcohol, pullulan, etc.

Besides them, sorbic acid; benzalconium chloride; cetylpyridiniumchloride; benzethonium chloride; parabens such as methylparaoxybenzoate, ethyl paraoxybezoate, propyl paraoxybenzoate, butylparaoxybenzoate, and others; gum acacia; sorbitol; magnesium stearate;talc; silica; microcrystalline cellulose; starch; calcium phosphate;vegetable oil; carboxymethylcellulose; sodium lauryl sulfate; water;ethanol; glycerin; and syrup.

Typical examples of surfactants are listed below. Among these, single orcombination of more than two of these surfactants can be added to theformulation in the invention.

Nonionic surfactants may include sorbitan esters of fatty acids, forexample, sorbitan monocaprilate, sorbitan monolaurate, sorbitanmonopalmitate, etc, and glycerol esters of fatty acids, for example,glyceryl monocaprilate, glyceryl monomyristate, glyceryl monostearate,etc, and polyglycerol esters of fatty acids, for example, decaglycerylmonostearate, decaglyceryl distearate, decaglyceryl monolinoleate, etc,and polyoxyethylene sorbitan esters of fatty acids, for example,polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitanmonooleate, polyoxyethylene sorbitan monostearate, polyoxyethylenesorbitan monopalmitate, polyoxyethylene sorbitan trioleate,polyoxyethylene sorbitan tristearate, etc, and polyoxyethylene sobitolesters of fatty acids, for example, polyoxyethylene sobitoltetrastearate, polyoxyethylene sobitol tetralaurate, etc, andpolyoxyethylene glycerol esters of fatty acids such as polyoxyethyleneglyceryl monostearate, and polyethylene glycerol esters of fatty acidssuch as polyethylene glyceryl distearate, and polyoxyethylene alkylether such as polyoxyethylene lauyl ether, and polyoxyethylenepolyoxypropylene alkyl ether, for example, polyoxyethylenepolyoxypropylene glycol ether, polyoxyethylene polyoxypropylenepropylether, polyoxyethylene polyoxypropylene cetyl ether, etc, andpolyoxyethylene alkylphenyl ether such as polyoxyethylene nonylphenylether, and polyoxyethylene caster oils, for example, polyoxyethylenecaster oil, polyoxyethylene hydrogenated caster oil, and polyoxyethyleneyellow beeswax derivatives such as polyoxyethylene sorbitol yellowbeeswax, and polyoxyethylene lanolin derivatives such as polyoxyethylenelanolin, and polyoxyethylene amide of fatty acids with HLB 6 to 18 suchas polyoxyethylene stearylamide.

Anionic surfactants may include alkyl sulfate (C₁₀ to C₁₈) salts, forexample, sodium cetyl sulfate, sodium lauryl sulfate, sodiumoleylsulfate, etc, and polyoxyethylene alkylether sulfate salts whoseaverage moles of added ethyleneoxide is 2 to 4 and carbons of alkylgroups is 10 to 18, such as sodium polyoxyethylene lauryl ether sulfate,and alkyl sulfo succinate ester salts whose length of alkyl groups is 8to 18 such as sodium lauryl sulfo succinic acid ester.

Naturally occurring sulfactants may include lecithin, and glycerol lipidphosphate, and sphingolipids such as sphingomyelin, and sucrose estersof fatty acids (C₁₂ to C₁₈).

Sulfur-containing reducing agents may include N-acety cysteine, N-acetyhomocysteine, thioctic acid, thioethanol, thioethanolamine,thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodiumthiosulfate, glutathione, thioalkanic acids (C₁ to C₇) having sulfhydrylgroup.

Anti-oxidants may include erysorbic acid, dibutylhydroxytoluene,butylhydroxyanisole, alpha-tocopherol, tocopherol acetate, L-ascorbicacid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodiumbisulfite, sodium sulfite, triamyl gallate, propyl gallate, andchelating agents, for example, calcium disodium edetate (EDTA), sodiumpyrophospate, sodium metaphosphate, etc.

For a pharmaceuticl composition of this invention, hPTH may exist atabout 0.01-20%, preferably at about 0.05-10%, and an organic acid may beadded as appropriate. The content of the latter prior to use is about0.05-99.5%, preferably about 0.1-99.0%. A carrier or excipient which isusually added during preparation of a medicinal product may be added asappropriate, or may exist, for example, at about 0.01-99.5% prior touse. Other various functional components may be added as appropriate, ormay exist, for example, at about 0.05-99.5% prior to use.

Preparation of the pharmaceutical composition for intranasaladministration of this invention may be achieved by any known method.

For example, an hPTH-based pharamceutical component in which the aceticacid content has been reduced may be served as a pharmaceuticalcomposition. Alternatively, to an hPTH-based pharmaceutical component inwhich the acetic acid content has been reduced, may be added asappropriate a carrier or excipient which is usually added duringpreparation of a pharmaceutical product, and an organic acid and othervarious functional components, and the resulting compound may be used asa pharmaceutical component. Addition of an organic acid may take placeto replace acetic acid, or simply for addition. For example, an hPTHpharmaceutical component to which are added as appropriate a carrier orexcipient which is usually added during preparation of a pharmaceuticalpreparation, an organic acid, and various functional components; aresulting mixture is dissolved for one time in distilled water; thesolution is lyophilized; and a uniform composition is thereby obtained.

Alternatively, an hPTH pharmaceutical component, and a carrier orexcipient which is usually used during preparation of a pharmaceuticalpreparation are dissolved for one time in distilled water; an organicacid and various functional components are then added to the solution;the resultant solution is lyophilized; and a uniform composition isthereby obtained. As a further variant, an hPTH pharmaceuticalcomponent, an organic acid, and various functional components aredissolved for one time in distilled water; the solution is lyophilized;a desired amount of the lyophilized compound is dissolved as needed incombination with a carrier or excipient which is usually used duringpreparation of a pharmaceutical preparation; and a uniform compositionis thereby obtained.

The pharmaceutical component of this invention may take various dosageforms depending on its expected administration routes: it may take aform appropriate for being applied to the mucosa of the rectum, nasalcavity, oral cavity, etc. The pharmaceutical composition for intranasaladministration of this invention is preferably applied in a formappropriate for intranasal use.

A preferred example of the pharmaceutical composition for intranasaladministration of this invention may occur as an on-demand dissolvableform of which a lyophilized portion contains a pharmaceuticalcomposition of this invention provided lyophilized and a dissolvingsolution portion is attached to the former.

An organic acid such as citric acid, adipic acid or glycolic acid whichis added for promoting absorption may exist as the constituent of a saltof hPTH, an adherent, or an additive. Alternatively, the organic acidmay be dissolved in the dissolving solution portion.

Administration of a pharmaceutical composition for intranasaladministration of this invention may be achieved by any known method.For example, spraying a pharmaceutical composition for intranasaladministration of this invention is applicable: the composition may beplaced in a container; a nebulizer is attached to the container; the tipof nozzle is inserted into the nasal cavity; and the pharmaceuticalcomposition is sprayed.

The dose of a pharmaceutical composition of this invention may varydepending on the kind of disease, the age and weight of the patient, theseverity of disease, and the route through which the composition isadministered. If, for example, an hPTH-based composition is appliednasally, it may be applied once daily or several times daily with eachdose reduced in proportion, successively for a period. A single dose ofhPTH(1-34)-based composition preferably occurs in the range of 10-5,000μg. During treatment, a so-called wash-out may be inserted, andtreatment may then be resumed.

EXAMPLE

The present invention will be detailed below by means of Examples, butthis invention should not be limited to those examples.

The testing methods and apparatuses used in Examples are based on whatis described below, unless otherwise stated.

1. Analysis of hPTH by HPLC

Determination of the content of a studied peptide in a composition, andchecking whether any decomposition products (by-products) are present inthe composition was achieved by reverse phase HPLC using the apparatusesand conditions specified below.

-   Apparatus: LC-9A system from Shimadzu Ltd.-   Column: YMC Protein-RP (4.6 mmø150 mm)-   Column temperature: 40° C.-   Eluate: the concentration of acetonitrile in 0.1% trifluoroacetic    acid is linearly varied from 25% to 40% in 30 minutes.-   Flow rate: 1 mL/min-   Detection: UV(210 nm)-   Injection amount: 50 μL    2. Analysis of Acetic Acid

The contents of acetic acid of dialysis solutions and of lyophilizedcompositions were determined by ion exchange chromatography under theconditions as specified below.

-   Apparatus: LC-91 system from Shimadzu Ltd.-   Column: IC-Al from Shimadzu (4.6 mmø×100 mm)-   Column temperature: 40° C.-   Eluate: 1:1 mixture of 0.84% aqueous solution of phthalic acid and    0.58% aqueous solution of tris hydroxymethyl aminomethane-   Flow rate: 1.5 mL/min-   Detection: electric conductivity detector-   Injection amount: 10 μL    3. Mass Analysis

Determination of the masses of hPTH, decomposition products(by-products) of hPTH, and their enzymatic digests was achieved with theapparatuses under conditions as specified below.

-   Apparatus: MAT TSQMS from Finnigan-   Ion source: ESI-   Detection mode: positive-   Spray voltage: 4.5 kV-   Capillary temperature: 250° C.-   Mobile phase: (1:1) mixture of 0.2% acetic acid and methanol-   Flow rate: 0.2 mL/min-   Scan range: m/z 550 - 850    4. Sequencing Amino Acids

Determination of the amino acid sequences of decomposition products(by-products) of hPTH and their enzymatic digests was achieved with thefollowing apparatuses.

-   Apparatus: type 477A sequencer from PerkinElmer    5. Determination of Amino Acid Composition

Determination of the amino acid compositions of hPTH, decompositionproducts (by-products) of hPTH, and their enzymatic digests was achievedwith the following apparatuses.

-   Apparatus: type L-8500 amino acid analyzer from Hitachi    6. Storage of Specimens (Stability Test)

The test specimens were stored in a depository kept under the conditionsas specified below.

-   Apparatus: LH-30-14 from Nagano Science Co. Ltd.-   Temperatures set: 1) 40±1° C., 2) 60+1° C., 3) 80±2° C.    7. Lyophilization-   Apparatus: RL-903BS from Kyowa Vacuum Engineering, Ltd.-   Vial: 15 mL glass vial

Reference Example 1

Production of hPTH(1-34) (1)

The expression plasmid pG117S4HPPH34 (Japanese Patent Laid-Open No.9-29660) containing a gene coding for a chimera protein of hPTH(1-34)obtained by connecting a DNA fragment coding for a derivative ofβ-galactosidase derived from E. coli with a DNA fragment coding forhPTH(1-34) through the intervention of a DNA fragment coding for alinker containing a cleavage motive (Lys-Arg) of Kex2 protease or aprocessing enzyme, was introduced into the cells of M25 strain E. coli(w3110/ompT: Sugimura et al., Biochem. Biophys. Res. Commun., vol. 153,1988, p. 753-759). The transformed cells of M25 strain E. coli werecultivated on a medium containing 2% yeast extract in a 20 L culturetank.

The cultivation was continued until the density of cells becameOD₆₆₀=12. The recovered cells were broken to pieces with a high pressurehomogenizer (Manton-Gaullin) in 10 mM Tris-HCl buffer (pH8.2)supplemented with 1 mM EDTA, centrifuged, and washed, to produce about625 mL of suspension containing about 100 g of inclusion bodies filledwith the chimera protein. To 250 mL of suspension containing 40 g ofinclusion bodies, were added 100 mL of 1M Tris-HCl buffer (pH8.2), 50 mLof 5M NaCl, 500 mL of deionized water, and 900 g of urea, and themixture was agitated at 30° C. to allow the inclusion bodies todissolve.

The solution was diluted with deionized water to 5 L, to which was added50 mL of 250 mM CaCl₂. Then, to the solution was added Kex2-660comprising amino acid residues designated an amino acid Nos. 1 - 660(Japanese Patent Laid-Open No. 10-229884) which is a derivative fromKex2 protease, until it existed at 20 kU/mL or higher. The mixture wasgently stirred for two hours, and hPTH(1-34) was cleaved from thechimera protein. The reaction solution was adjusted to pH6.4 withaddition of acetic acid; it was then diluted two fold with deionizedwater, thereby allowing the chimera protein and γ-galactosidasederivative remaining unreacted to precipitate; and the yield wascentrifuged to give a supernatant containing 6.7 g of hPTH(1-34). Thesupernatant was adjusted to pH5.0 with addition of acetic acid; thesolution was applied to a cation exchange resin (SP Toyopearl from TosohCorporation) previously equilibrated with 10 mM sodium acetate to allowhPTH(1-34) to be adsorbed to the resin; the resin was washed with 10 mMsodium acetate buffer; and 0.4M NaCl was used to give a fractioncontaining 6.0 g of hPTH(1-34).

To this fraction was added acetic acid to 3 v/v %; the solution wasapplied to a column for low pressure reverse phase ODS (Soken ODS-W fromSoken Chemicals Co.) previously equilibrated with 3 v/v % acetic acid;and 30 v/v % acetonitrile containing 3 v/v % acetic acid was used toeluate hPTH(1-34). The eluate containing hPTH(1-34) was enriched under areduced pressure; the yield was applied to a column for reverse phaseHPLC (TSKge10DS120T with a size of 55 mm×600 mm from Tosoh Corp.); andsolution of acetonitrile in 5 v/v % acetic acid was allowed to flow at40 mL/min for 60 minutes with the concentration of acetonitrile beinglinearly varied from 16% to 32% in the mean time, to eluate hPTH(1-34).Thus, a purified fraction containing 4 g of hPTH(1-34) was obtained.

A 60 g of the remaining inclusion bodies was similarly treated, andanother purified fraction containing 5 g of hPTH(1-34) obtainedtherefrom was combined with the former; the mixture was removed ofacetonitrile under a reduced pressure; and the yield was diluted with 5v/v % acetic acid such that the concentration of hPTH(1-34) fell to 10mg/mL. A 15 ml of the solution was placed in each glass vial; and allthe vials containing the solution were lyophilized to give 9 g ofhPTH(1-34) in total (150 mg×60 vials).

ESI-MS: 4117.7 (theoretical value being 4117.8). Amino acid compositionafter being hydrolyzed with 6N hydrochloric acid: Asx-4.0(4);Ser-2.6(3); Glx-4.9(5); Gly-1.0(1); Val-3.0(3), Met-2.0(2); Ile-1.0(1);Leu-5; Phe-1.1(1); Lys-30(3); His-3.0(3); Arg-2.0(2); and Trp-notdetected (1).

Reference Example 2

Production of hPTH(1-34) (2)

Similar live microbes to those used in Reference Example 1 werecultivated in a 200 L culture tank. The cultivation was continued untilthe density of cells became OD₆₆₀=160. The recovered cells were brokento pieces with a high pressure homogenizer in 10 mM Tris-HCl buffer(pH8.2) supplemented with 1 mM EDTA, centrifuged, and washed, to produceabout 10 L of suspension containing about 5 kg of inclusion bodiesfilled with the chimera protein.

To 4.0 L of suspension containing 2 kg of inclusion bodies, were added1.6 L of 1M Tris-HCl buffer (pH8.2), 0.8 L of 5M NaCl, 15 L of deionizedwater, and 13 kg of urea, and the mixture was agitated at 30° C. toallow the inclusion bodies to dissolve.

The solution was diluted with deionized water to 80 L, to which wasadded 0.8 mL of 250 mM CaCl₂. Then, to the solution was added Kex2-660(Japanese Patent Laid-Open No. 10-229884), until it existed at 10 kU/mLor higher. The mixture was gently agitated for one hour, and hPTH(1-34)was separated by cleavage from the chimera protein. The reactionsolution was adjusted to pH6.3 with addition of acetic acid; it was thendiluted two fold with deionized water, thereby allowing the chimeraprotein and β-galactosidase derivative remaining unreacted toprecipitate; and the yield was subjected to pressurized filtration togive a supernatant containing hPTH(1-34).

The supernatant was adjusted to pH5.0 with addition of acetic acid; thesolution was applied to a cation exchange resin column (5 L)(Poros 50HSfrom PerSeptive Biosystems, USA) previously equilibrated with 10 mMsodium acetate buffer to allow hPTH(1-34) to be adsorbed to the column;the column was washed with 10 mM sodium acetate buffer; and 0.4M NaClwith a concentration gradient was used to give a fraction containinghPTH(1-34). To this fraction was added acetic acid to 3 v/v %; thesolution was applied to a column (5 L) for low pressure reverse phaseODS (Soken ODS-W from Soken Chemicals Co.) previously equilibrated with3 v/v % acetic acid; and 30 v/v % acetonitrile containing 3 v/v % aceticacid was used to eluate hPTH(1-34).

The eluate containing hPTH(1-34) was enriched under a reduced pressure;the yield was filtrated through a 0.22 μm filter; the filtrate wasapplied to a column for reverse phase HPLC (TSK ODS 80Ts 20 μm, 105mmID×550 mm from Tosoh Corp.); and acetonitirile with a concentrationgradient was used in the presence of 3 v/v % acetic acid to eluatehPTH(1-34). Several eluates thus obtained were combined; and thesolution was removed of acetonitrile through distillation under areduced pressure, to give 10.4 L of conc. hPTH(1-34) solution containing70 g of hPTH(1-34) at 99.6%. Out of 3 kg of the remaining inclusionbodies, 2 kg was subjected to the same purification process, to give11.6 L of conc. hPTH(1-34) solution containing hPTH(1-34) at 99.6%.

Reference Example 3

Production of hPTH(1-84)

The expression plasmid pGP#19 (Japanese Patent Laid-Open No. 9-29660)containing a gene coding for a chimera protein of hPTH(1-84) obtained byconnecting a DNA fragment coding for a derivative of β-galactosidasederived from E. coli with a DNA fragment coding for hPTH(1-84) throughthe intervention of a DNA fragment coding for a linker containing acleavage motive (Lys-Arg) of Kex2 protease or a processing enzyme, wasintroduced into the cells of M25 strain E. coli. The transformed cellsof M25 strain E. coli were cultivated at 37° C. in a 3 L culture tank.The cultivation was continued until the turbidity (OD₆₆₀) of culturesolution became OD₆₆₀=1. Then, isopropyl beta-thiogalactoside (IPTG) wasadded to 1.0 mM.

The cultivation was further continued for four hours. The cells wererecovered by centrifugation; and the cells were then suspended in TE (10mM Tris and 1 mM EDTA at pH8.0). The cells were broken to pieces with aFrench press, subjected to a repetition of centrifugation andresuspension, and washed, to produce inclusion bodies. A suspension ofthe inclusion bodies was added for dissolution to a solution (pH8.0)containing 8.0M urea and 10 mM Tris, and centrifuged; the supernatantwas loaded onto a Toyopearl column (Toso Corp.); and NaCl with aconcentration gradient of 0-0.4M was used to eluate enriched hPTH(1-84).The inclusion body dissolving enriched solution was further concentratedby ultrafiltration with a removal limit of 10,000 MW. The yield wasdiluted to give a solution in which the constituents became 50 mM forBisTris at pH6.8, 1.0 mM for CaCl₂, and 5 mg/mL for the chimera protein.Kex2-660 was added to 2 kU/mL, and reaction was allowed to proceed at30° C. for one hour to separate hPTH(1-84) by cleavage.

The reaction solution was adjusted to pH5.0 with addition of aceticacid; it was then diluted two fold with deionized water, therebyallowing the chimera protein and β-galactosidase derivative remainingunreacted to precipitate; and the yield was centrifuged to give asupernatant containing hPTH(1-84). To the supernatant was added aceticacid to 3 v/v %; and the solution was applied to TSK gel ODS 80Ts (21mmID×250 mm, Tosoh Corporation) previously equilibrated with 3 v/v %acetic acid for purification. Fractions containing 98% or higherhPTH(1-84) were combined; the resulting solution was removed of solvent;and the residue was lyophilized to give 500 mg of hPTH(1-84). Themolecular weight and amino acid composition of this substance are asindicated below. Based on those data, the substance was identified ashPTH(1-84).

ESI-MS: 9424.7 (theoretical value being 9424.7). Amino acid compositionafter being hydrolyzed with 6N hydrochloric acid: Asx-10.1(10);Thr-1.1(1); Ser-6.3(7); Glx-11.0(11); Pro-2.9(3); Gly-4.1(4);Ala-7.0(7); Val-8.0(8); Met-1.9(2); Ile-1.0(1); Leu-10; Phe-1.0(1);Lys-8.9(9); His-3.9(4); Arg-5.0(5); and Trp-not detected (1).

Example 1

Removal of Acetic Acid from hPTH(1-34) (1)

(1) An amount of preparation corresponding to 150 mg as hPTH(1-34)obtained in Reference Example 1 was dissolved in distilled water (30mL), to give an aqueous solution (pH4.7) of hPTH(1-34) at 5 mg/mL. Thissolution was dialyzed against distilled water (100 mL) with a GI microacilyzer (Asahi Kasei Corp.) incorporating an electrodialysis membraneAC-130-10 (Asahi Kasei Corp.). The dialysis was continued for removal ofacetic acid until the pH of the dialysis solution became pH5.0. Theenriched solution containing 6.8% acetic acid was lyophilized, and driedmass corresponding to about 150 mg as hPTH(1-34) was obtained as aceticacid content being 4.8%.

(2) An amount of preparation corresponding to 150 mg as hPTH(1-34)obtained in Reference Example 1 was dissolved in distilled water (30mL), to give an aqueous solution (pH4.7) of hPTH(1-34) at 5 mg/mL. Thissolution was subjected to electrodialysis as in Example (1): thedialysis was continued for removal of acetic acid until the pH of thedialysis solution became pH5.5. The enriched solution containing 4.6%acetic acid was lyophilized, and dried mass corresponding to about 150mg as hPTH(1-34) was obtained as acetic acid content being 3.8%.

(3) An amount of preparation corresponding to 150 mg as hPTH(1-34)obtained in Reference Example 1 was dissolved in distilled water (30mL), to give an aqueous solution (pH4.7) of hPTH(1-34) at 5 mg/mL. Thissolution was subjected to electrodialysis as in Example (1): thedialysis was continued for removal of acetic acid until the pH of thedialysis solution became pH5.9. The enriched solution containing 3.1%acetic acid was lyophilized, and dried mass corresponding to about 150mg as hPTH(1-34) was obtained as acetic acid content being 2.9%. (4) Anamount of preparation corresponding to 150 mg as hPTH(1-34) obtained inReference Example 1 was dissolved in distilled water (30 mL), to give anaqueous solution (pH4.7) of hPTH(1-34) at 5 mg/mL. This solution wassubjected to electrodialysis as in Example (1): the dialysis wascontinued for removal of acetic acid until the pH of the dialysissolution became pH7.0. The enriched solution containing 1.6% acetic acidwas lyophilized, and dried mass corresponding to about 150 mg ashPTH(1-34) was obtained as acetic acid content being 1.6%.

From above results it was demonstrated that with the reduction of aceticacid content, the decrement of acetic acid content by lyophilizationbecomes smaller, and that with the reduction of acetic acid content, thefractional decrement of acetic acid content becomes smaller.Accordingly, reduction of the content of acetic acid in an hPTHcomponent is useful for preparing a pharmaceutical hPTH componentcontaining a specified amount of acetic acid, which has been difficultto obtain by conventional techniques.

Example 2

Removal of Acetic Acid from hPTH(1-34) (2)

To a solution containing preparation corresponding to about 150 mg ashPTH(1-34) obtained in Reference Example 2 was added 5N NaOH to pH5.5.One fourth of the solution was loaded onto a column for low pressurereverse phase ODS (Soken ODS-W (800 mL) from Soken Chemicals Co.)previously equilibrated with 5 v/v % aqueous solution of acetonitrile,to allow hPTH(1-34) to be adsorbed to the column. A 4 L of 5 v/v %aqueous solution of acetonitrile was flowed to eluate sodium acetate;and 50 v/v % aqueous solution of acetonitrile was then flowed to eluatehPTH(1-34). This process was repeated for each of the four parts; thehPTH(1-34) rich fractions of the four parts were combined to give 8.2 Lof nearly acetic acid free fraction containing 122.5 g of PTH(1-34) wasobtained (1.13% acetic acid content to hPTH(1-34)).

To this fraction was added distilled water to give an hPTH(1-34)solution at 10 mg/mL; to this solution was added 2.3 g of acetic acidsuch that the resulting solution contains 122.5 g of PTH(1-34) with 3%acetic acid content at 3%; and the mixture was well stirred. Thesolution was distributed to vials such that each vial contains 150 mg ofhPTH(1-34); and the hPTH(1-34) solution in vial was lyophilized, whichgave hPTH(1-34) as acetic acid content being 2.1%.

Example 3.

Removal of Acetic Acid from hPTH(1-34) (3)

(1) An amount of preparation corresponding to 300 mg as hPTH(1-34)obtained in Reference Example 1 was dissolved in distilled water (30mL), to give an aqueous solution of hPTH(1-34) at 10 mg/mL. Thissolution (pH4.7) containing 9.5% acetic acid was placed in three glassvials, 2 mL for each vial; and they were placed in an FZ-6 lyophilizer(Laboconco Corp.) whose internal pressure was kept at 0.13 mBar orlower; and they were subjected to primary drying (−20° C. in terms ofthe temperature of the shelf on which they rested for 12 hours) andsecondary drying (25° C. at the shelf for 48 hours) for lyophilization.On completion of the secondary drying, the chamber used forlyophilization was filled with nitrogen gas; and the vials wereautomatically capped. A 2 mL of distilled water was introduced into eachvial, and the solution was determined by ion exchange HPLC of itscontent of acetic acid. The results are shown in Table 1.

(2) An amount of preparation corresponding to 300 mg as hPTH(1-34)obtained in Reference Example 1 was dissolved in distilled water (30mL), to give an aqueous solution of hPTH(1-34) at 10 mg/mL. Thissolution (pH4.7) was subjected to electrodialysis using a micro acilyzer(Asahi Kasei Corp.) incorporating an electrodialysis membrane AC-130-10(Asahi Kasei Corp.). The dialysis was continued for removal of aceticacid until the pH of the dialysis solution became pH5.0 (the content ofacetic acid of the solution subject to dialysis being 7.3%). The aceticacid-removed solution was placed in three glass vials, 2 mL for eachvial; they were lyophilized as in Example (1); and the content of aceticacid in each sample was determined by ion exchange HPLC. The results areshown in Table 1.

(3) An amount of preparation corresponding to 300 mg as hPTH(1-34)obtained in Reference Example 1 was dissolved in distilled water (30mL), to give an aqueous solution of hPTH(1-34) at 10 mg/mL. Thissolution (pH4.7) was subjected to electrodialysis using a micro acilyzer(Asahi Kasei Corp.) incorporating an electrodialysis membrane AC-130-10(Asahi Kasei Corp.). The dialysis was continued for removal of aceticacid until the pH of the dialysis solution became pH5.5 (the content ofacetic acid of the solution subject to dialysis being 4.6%). The aceticacid-removed solution was placed in three glass vials, 2 mL for eachvial; they were lyophilized as in Example (1); and the content of aceticacid in each sample was determined by ion exchange HPLC. The results areshown in Table 1.

(4) An amount of preparation corresponding to 300 mg as hPTH(1-34)obtained in Reference Example 1 was dissolved in distilled water (30mL), to give an aqueous solution of hPTH(1-34) at 10 mg/mL. Thissolution (pH4.7) was subjected to electrodialysis using a micro acilyzer(Asahi Kasei Corp.) incorporating an electrodialysis membrane AC-130-10(Asahi Kasei Corp.). The dialysis was continued for removal of aceticacid until the pH of the dialysis solution became pH6.3 (the content ofacetic acid of the solution subject to dialysis being 2.0%). The aceticacid-removed solution was placed in three glass vials, 2 mL for eachvial; they were lyophilized as in Example (1); and the content of aceticacid in each sample was determined by ion exchange HPLC. The results areshown in Table 1.

(5) An amount of preparation corresponding to 300 mg as hPTH(1-34)obtained in Reference Example 1 was dissolved in distilled water (30mL), to give an aqueous solution of hPTH(1-34) at 10 mg/mL. Thissolution (pH4.7) was subjected to electrodialysis using a micro acilyzer(Asahi Kasei Corp.) incorporating an electrodialysis membrane AC-130-10(Asahi Kasei Corp.). The dialysis was continued for removal of aceticacid until the pH of the dialysis solution became pH7.0 (the content ofacetic acid of the solution subject to dialysis being 1.1%). The aceticacid-removed solution was placed in three glass vials, 2 mL for eachvial; they were lyophilized as in Example (1); and the content of aceticacid in each sample was determined by ion exchange HPLC. The results areshown in Table 1.

(6) An amount of preparation corresponding to 300 mg as hPTH(1-34)obtained in Reference Example 1 was dissolved in distilled water (30mL), to give an aqueous solution of hPTH(1-34) at 10 mg/mL. Thissolution (pH4.7) was subjected to electrodialysis using a micro acilyzer(Asahi Kasei Corp.) incorporating an electrodialysis membrane AC-130-10(Asahi Kasei Corp.). The dialysis was continued for removal of aceticacid until the pH of the dialysis solution became pH7.6 (the content ofacetic acid of the solution subject to dialysis being 0.5%). The aceticacid-removed solution was placed in three glass vials, 2 mL for eachvial; they were lyophilized as in Example (1); and the content of aceticacid in each sample was determined by ion exchange HPLC. The results areshown in Table 1.

As is obvious from Table 1, with the reduction of acetic acid content ofa sample prior to lyophilization, the decrement of acetic acid contentby lyophilization becomes smaller, and that with the reduction of aceticacid contents of samples prior to lyophilization, the variation inacetic acid content among the samples or lots after lyophilizationbecomes smaller. Accordingly, hPTH with low acetic acid content isdemonstrated to be useful for preparing a pharmaceutical hPTH componentcontaining a specified amount of acetic acid, which has been difficultto obtain by conventional techniques. TABLE 1 Change of acetic acidcontents as a result of lyophilization Difference of Acetic acid Aceticacid acetic acid Dialysis content before content after contents beforesolution lyophilization lyophilization Average and after pH (%) (%) (%)lyophilization (%) 4.7 9.5 6.7 6.6 2.9 6.5 6.6 5.0 7.3 5.5 5.5 1.8 5.45.5 5.5 4.6 4.9 4.9 −0.3 4.9 4.8 6.3 2.0 2.1 2.1 −0.1 2.1 2.1 7.0 1.10.8 0.8 0.3 0.8 0.8 7.6 0.5 0.4 0.4 0.1 0.4 0.5

Example 4

Removal of Acetic Acid from hPTH(1-34)

(1) Similar live microbes to those used in Reference Example 1 werecultivated in a 200 L culture tank. The cells were broken to pieces,centrifuged, and washed; and 4.8 kg of inclusion bodies filled with achimera protein was obtained. A 2.4 kg out of this was subjected todigestion by Kex2-660 as in Reference Example 2 to obtain hPTH(1-34).The yield was then subjected to purification by cation exchangechromatography, desaltation by reverse phase ODS chromatography, andfinal purification by reverse phase HPLC. Then, as in Example 2, to thepurified fraction was added 5N sodium hydroxide to pH5.5; and thesolution was loaded onto a low pressure reverse phase ODS column. A 5v/v % aqueous solution of acetonitrile was passed to remove sodiumacetate; and 50 v/v % aqueous solution of acetonitrile was then flowedto eluate hPTH(1-34). Thus, 5.4 L of solution containing 58 g ofPTH(1-34) was obtained (1.2% acetic acid content to hPTH1(1-34). To thissolution was added 1.0 g of acetic acid such that the resulting solutioncontains 58 g of PTH(1-34) with 3% acetic acid content; and the mixturewas well stirred. The solution was distributed to vials such that eachvial contains 150 mg of hPTH(1-34); and all the hPTH(1-34) solutions invial were lyophilized, which gave 57.8 g of hPTH(1-34) as acetic acidcontent being 2.5% (385 vials).

(2) An amount of preparation corresponding to 150 mg as hPTH(1-34)obtained in Example (1) was dissolved in 3 mL of trifluoroethanol; and30 mL of diethylether was added for precipitation. A powder obtainedtherefrom was subjected to drying for 24 hours under a reduced pressurein a desiccator in the presence of sodium hydroxide pellets, to give 130mg of hPTH(1-34) as acetic acid content being 2.0%.

(3) Into the vial containing preparation corresponding to 150 mg ashPTH(1-34) obtained in Example (1) was introduced 1.53 μL of acetic acidalong the internal wall of the vial with a micro-syringe while carebeing taken not to contact it with hPTH(1-34). Vaporization of aceticacid was performed by placing the vial at 80° C. for five minutes; thevial was agitated with a vortex mixer; and hPTH(1-34) in the vial wasturned into fine powder, to give an hPTH(1-34) preparation as aceticacid content being 3.6%.

(4) Into the vial containing preparation corresponding to 150 mg ashPTH(1-34) obtained in Example (1) was introduced 3.78 μL of acetic acidalong the internal wall of the vial with a micro-syringe while carebeing taken not to contact it with hPTH(1-34). Vaporization of aceticacid was performed by placing the vial at 80° C. for five minutes; thevial was agitated with a vortex mixer; and hPTH(1-34) in the vial wasturned into fine powder, to give an hPTH(1-34) preparation as aceticacid content being 5.1%.

(5) Into the vial containing preparation corresponding to 150 mg ashPTH(1-34) obtained in Example (1) was introduced 5.28 μL of acetic acidalong the internal wall of the vial with a micro-syringe while carebeing taken not to contact it with hPTH(1-34). Vaporization of aceticacid was performed by placing the vial at 80° C. for five minutes; thevial was agitated with a vortex mixer; and hPTH(1-34) in the vial wasturned into fine powder, to give an hPTH(1-34) preparation as aceticacid content being 6.2%.

Reference Example 4

(1) Into the vial containing preparation corresponding to 150 mg ashPTH(1-34) obtained in Example 4(1) was introduced 7.23 μL of aceticacid along the internal wall of the vial with a micro-syringe while carebeing taken not to contact it with hPTH(1-34). Vaporization of aceticacid was performed by placing the vial at 80° C. for five minutes; thevial was agitated with a vortex mixer; and hPTH(1-34) in the vial wasturned into fine powder, to give an hPTH(1-34) preparation as aceticacid content being 7.5%.

(2) Into the vial containing preparation corresponding to 150 mg ashPTH(1-34) obtained in Example 4(1) was introduced 9.48 μL of aceticacid along the internal wall of the vial with a micro-syringe while carebeing taken not to contact it with hPTH(1-34). Vaporization of aceticacid was performed by placing the vial at 80° C. for five minutes; thevial was agitated with a vortex mixer; and hPTH(1-34) in the vial wasturned into fine powder, to give an hPTH(1-34) preparation as aceticacid content being 9.1%.

(3) Into the vial containing preparation corresponding to 150 mg ashPTH(1-34) obtained in Example 4(1) was introduced 10.53 μL of aceticacid along the internal wall of the vial with a micro-syringe while carebeing taken not to contact it with hPTH(1-34). Vaporization of aceticacid was performed by placing the vial at 80° C. for five minutes; thevial was agitated with a vortex mixer; and hPTH(1-34) in the vial wasturned into fine powder, to give an hPTH(1-34) preparation as aceticacid content being 7.5%.

(4) Into the vial containing preparation corresponding to 150 mg ashPTH(1-34) obtained in Example 4(1) was introduced 14.28 μL of aceticacid along the internal wall of the vial with a micro-syringe while carebeing taken not to contact it with hPTH(1-34). Vaporization of aceticacid was performed by placing the vial at 80° C. for five minutes; thevial was agitated with a vortex mixer; and hPTH(1-34) in the vial wasturned into fine powder, to give an hPTH(1-34) preparation as aceticacid content being 12.5%.

(5) Into the vial containing preparation corresponding to 150 mg ashPTH(1-34) obtained in Example 4(1) was introduced 16 μL of acetic acidalong the internal wall of the vial with a micro-syringe while carebeing taken not to contact it with hPTH(1-34). Vaporization of aceticacid was performed by placing the vial at 80° C. for five minutes; thevial was agitated with a vortex mixer; and hPTH(1-34) in the vial wasturned into fine powder, to give an hPTH(1-34) preparation as aceticacid content being 13.7%.

(6) Into the vial containing preparation corresponding to 150 mg ashPTH(1-34) obtained in Example 4(1) was introduced 22.6 μL of aceticacid along the internal wall of the vial with a micro-syringe while carebeing taken not to contact it with hPTH(1-34). Vaporization of aceticacid was performed by placing the vial at 80° C. for five minutes; thevial was agitated with a vortex mixer; and hPTH(1-34) in the vial wasturned into fine powder, to give an hPTH(1-34) preparation as aceticacid content being 18.3%.

(7) Into the vial containing preparation corresponding to 150 mg ashPTH(1-34) obtained in Example 4(1) was introduced 100 μL of acetic acidalong the internal wall of the vial with a micro-syringe while carebeing taken not to contact it with hPTH(1-34). Vaporization of aceticacid was performed by placing the vial at 80° C. for five minutes; thevial was agitated with a vortex mixer; and hPTH(1-34) in the vial wasturned into fine powder, to give an hPTH(1-34) preparation as aceticacid content being 72.5%.

Example 5

Removal of Acetic Acid from hPTH(1-84)

(1) An amount of preparation corresponding to 50 mg as hPTH(1-84)obtained as acetic acid content being 5.4% in Reference Example 3 wasdissolved in distilled water (10 mL). To give an aqueous solution(pH4.7) of hPTH(1-84) at 5 mg/mL. This solution was dialyzed at roomtemperature against distilled water (100 mL) with a GI micro acilyzer(Asahi Kasei Corp.) incorporating an electrodialysis membrane AC-130-10(Asahi Kasei Corp.). The dialysis was continued for removal of aceticacid until the pH of the dialysis solution became pH5.0. The enrichedsolution was lyophilized, and preparation corresponding to about 50 mgas hPTH(1-84) was obtained as acetic acid content being 3.9%.

(2) An amount of preparation corresponding to 50 mg as hPTH(1-84)obtained as acetic acid content being 5.4% in Reference Example 3 wasdissolved in distilled water (10 mL), to give an aqueous solution(pH4.7) of hPTH(1-84) at 5 mg/mL. This solution was subject toelectrodialysis in the same manner as in Example.(1). The dialysis wascontinued for removal of acetic acid until the pH of the dialysissolution became pH6.0. The enriched solution was lyophilized, andpreparation corresponding to about 50 mg as hPTH(1-84) was obtained asacetic acid content being 2.5%.

(3) An amount of preparation corresponding to 50 mg as hPTH(1-84)obtained as acetic acid content being 5.4% in Reference Example 3 wasdissolved in distilled water (10 mL), to give an aqueous solution(pH4.7) of hPTH(1-84) at 5 mg/mL. This solution was subjected toelectrodialysis in the same manner as in Example (1). The dialysis wascontinued for removal of acetic acid until the pH of the dialysissolution became pH7.0. The enriched solution was lyophilized, andpreparation corresponding to about 50 mg as hPTH(1-84) was obtained asacetic acid content being 1.3%.

(4) An amount of preparation corresponding to 50 mg as hPTH(1-84)obtained as acetic acid content being 5.4% in Reference Example 3 wasdissolved in distilled water (10 mL), to give an aqueous solution(pH4.7) of hPTH(1-84) at 5 mg/mL. This solution was subjected toelectrodialysis in the same manner as in Example (1). The dialysis wascontinued for removal of acetic acid until the pH of the dialysissolution became pH8.0. The enriched solution was lyophilized, andpreparation corresponding to about 50 mg as hPTH(1-84) was obtained asacetic acid content being 0.9%.

Example 6

Removal of Acetic Acid from hPTH(1-84) (2)

(1) An amount of preparation corresponding to 100 mg as hPTH(1-84)obtained as acetic acid content being 5.4% in Reference Example 3 wasdissolved in 500 μL of trifluoroethanol; and 20 mL of diethylether wasadded for precipitation. The precipitate was recovered by filtration;and the yield was subjected to drying for 24 hours under a reducedpressure in a desiccator in the presence of sodium hydroxide pellets, togive dried mass corresponding to about 90 mg as hPTH(1-84) obtained asacetic acid content being 1.6%.

(2) A 5.49 mg of dried mass obtained in Example 6(1) was preciselyweighed and placed in a 5 mL vial; and 0.3 μL of 10 vol % aceticacid/methylene chloride was added with a micro-syringe, to givepreparation as acetic acid content being 2.2%.

(3) A 5.58 mg of dried mass obtained in Example 6(1) was preciselyweighed and placed in a 5 mL vial; and 0.6 μL of 10 vol % aceticacid/methylene chloride was added with a micro-syringe, to givepreparation as acetic acid content being 2.7%.

(4) A 4.96 mg of dried mass obtained in Example 6(1) was preciselyweighed and placed in a 5 mL vial; and 1.0 μL of 10 vol % aceticacid/methylene chloride was added with a micro-syringe, to givepreparation as acetic acid content being 3.8%.

Reference Example 5

(1) A 5.05 mg of dried mass obtained in Example 6(1) was preciselyweighed and placed in a 5 mL vial; and 1.7 μL of 10 vol % aceticacid/methylene chloride was added with a micro-syringe, to givepreparation as acetic acid content being 5.2%.

(2) A 5.59 mg of dried mass obtained in Example 6(1) was preciselyweighed and placed in a 5 mL vial; and 2.5 μL of 10 vol % aceticacid/methylene chloride was added with a micro-syringe, to givepreparation as acetic acid content being 6.4%.

(3) A 5.01 mg of dried mass obtained in Example 6(1) was preciselyweighed and placed in a 5 mL vial; and 3.0 μL of 10 vol % aceticacid/methylene chloride was added with a micro-syringe, to givepreparation as acetic acid content being 8.0%.

(4) A 5.48 mg of dried mass obtained in Example 6(1) was preciselyweighed and placed in a 5 mL vial; and 4.4 μL of 10 vol % aceticacid/methylene chloride was added with a micro-syringe, to givepreparation as acetic acid content being 10.2%.

(5) A 5.47 mg of dried mass obtained in Example 6(1) was preciselyweighed and placed in a 5 mL vial; and 5.5 μL of 10 vol % aceticacid/methylene chloride was added with a micro-syringe, to givepreparation as acetic acid content being 12.3%.

(6) A 5.10 mg of dried mass obtained in Example 6(1) was preciselyweighed and placed in a 5 mL vial; and 10.2 μL of 10 vol % aceticacid/methylene chloride was added with a micro-syringe, to givepreparation as acetic acid content being 22.9%.

(7) A 5.53 mg of dried mass obtained in Example 6(1) was preciselyweighed and placed in a 5 mL vial; and 16.6 μL of 10 vol % aceticacid/methylene chloride was added with a micro-syringe, to givepreparation as acetic acid content being 33.6%.

Experiment 1

Stability of hPTH(1-34) (1)

The hPTH preparation from which acetic acid existing as the constituentof a salt of hPTH or an adherent, had been removed was tested for itsstability.

An amount of preparation corresponding to about 150 mg as hPTH(1-34)obtained as acetic acid content being 9.5% introduced into a glass vialand helmetically sealed as obtained in Reference Example 1 was stored inan LH-30-14 depository (Nagano Science Co.) kept at 40±1° C. for sixmonths. Prior to storage, some vials and after storage the remainingvials were subjected to reverse phase HPLC, thereby isolatingdecomposition products (by-products) prior to storage as well assubsequent to storage for their structural analysis. The results areshown in Table 2.

The reverse phase HPLC chromatogram of the sample having undergonestorage is shown in FIG. 1. A high peak representing hPTH(1-34) wasfollowed by three peaks (retention time being 13 to 17 minutes)designated as B, C and D. The percent areas of those peaks are 3.9% forpeak B, 6.9% for peak C, and 3.0% for peak D as indicated in Table 2.The total is 13.8% or a considerable fraction which forms a major causefor the deterioration of the product. Structural analysis was introducedto identify the compounds responsible for respective peaks anddemonstrated peak B is represented by a mixture of[Nε-acetyl-Lys¹³]-hPTH(1-34) and [Nε-acetyl-Lys²⁶]-hPTH(1-34), peak C by[Nα-acetyl-Ser¹]-hPTH(1-34), and peak D by [Nε-acetyl-Lys²⁷]-hPTH(1-34).

Next, the hPTH(1-34) preparation as acetic acid content being 2.9% asobtained in Example 1(3) was studied for its stability in the samemanner as above.

An amount of preparation corresponding to about 150 mg as hPTH(1-34)obtained as acetic acid content being 2.9% introduced into a glass vialand helmetically sealed was stored in an LH-30-14 depository (NaganoScience Co.) kept at 40±1° C. for six months. Prior to storage, somevials and after storage the remaining vials were subjected to reversephase HPLC, thereby isolating decomposition products (by-products) priorto storage as well as subsequent to storage for their structuralanalysis. The results are shown in Table 3.

The reverse phase HPLC chromatogram of the sample having undergonestorage is shown in FIG. 2. As is obvious from FIG. 2, when it iscompared with the one from the hPTH(1-34) as acetic acid content being9.5%, all the areas of peaks B, C and D are reduced. The areas of peaksB, C and D are in total 0.2% which is similar to the corresponding valueobserved in the hPTH(1-34) as acetic acid content being 9.5%. Thepost-storage value of the total peak areas here concerned is 3.6%, whichis far below the corresponding value (13.8%) of the hPTH(1-34) as aceticacid content being 9.5%.

It was demonstrated from above that acetyl bodies are majordecomposition products derived from hPTH, and that reduction of thecontent of acetic acid existing as the constituent of a salt ofhPTH(1-34) or an adherent will lead to an improved stability of thehPTH. Namely, reduction of the acetic acid content of an hPTHpreparation will lead to the production of an hPTH-based pharmaceuticalcomponent having an excellent stability. TABLE 2 Stability of hPTH(1-34)(acetic acid content: 9.5%, storage at 40° C. for 6 months) Afterstorage Decomposition products Before storage (peak area Peak(by-products) (peak area %) %) X1 [Met(O)⁸]-hPTH(1-34) 0.1% or less 0.1%X2 [Met(O)¹⁸]-hPTH(1-34) 0.1% or less 0.1% B* [Nε-AcLys¹⁸]-hPTH(1-34)Not detected 3.9% [Nε-AcLys²⁶]-hPTH(1-34) C [Nα-AcSer¹]-hPTH(1-34)  0.2%6.9% D [Nε-AcLys²⁷]-hPTH(1-34) Not detected 3.0% Others Unidentified 0.1% 4.9% decomposition products hPTH(1-34) 99.6% 81.1% Total 100.0% 100.0%*Peak B represents a mixture.

TABLE 3 Stability of hPTH(1-34) (acetic acid content: 2.9%, storage at40° C. for 6 months) After storage Decomposition products Before storage(peak area Peak (by-products) (peak area %) %) X1 [Met(O)⁸]-hPTH(1-34) 0.1% 0.1% X2 [Met(O)¹⁸]-hPTH(1-34)  0.1% 0.1% B*[Nε-AcLys¹⁸]-hPTH(1-34) Not detected 1.4% [Nε-AcLys²⁶]-hPTH(1-34) C[Nα-AcSer¹]-hPTH(1-34)  0.2% 1.0% D [Nε-AcLys²⁷]-hPTH(1-34) Not detected1.2% Others Unidentified  0.1% 3.9% decomposition products hPTH(1-34)99.5% 92.3% Total 100.0%  100.0%*Peak B represents a mixture.

Experiment 2

Stability of hPTH(1-34) (2)

Stored at 80° C. for 15 hours were hPTH(1-34) preparations as obtainedin Example 4 and Reference Example 4 which contained the respectivecontents of acetic acid contents as specified in Example 4 and ReferenceExample 4; to each of the preparations was added 15 mL of distilledwater with a syringe; and for each preparation, its content ofdecomposition products (by-products) was determined before and afterstorage. The results are shown in Table 4. The content (%) of acetylbodies (B, C and D) of each hPTH(1-34) preparation after storage isplotted as a function of its acetic acid content in FIG. 3.

As is obvious from Table 4 and FIG. 3, with the reduction of acetic acidcontent, decomposition products B, C and D derived from acetyl bodiesdecrease.

The purity of hPTH(1-34) preparations was plotted as a function of theiracetic acid contents in FIG. 4. As is obvious from FIG. 4, the curvetakes a sigmoid course with a deflection point at the chemicalequivalent (acetic acid content equal to about 7.3%) and if the aceticacid content is kept below the chemical equivalent, the stability of theproduct is rapidly improved.

Namely, reduction of the acetic acid content of an hPTH preparation willlead to the production of an hPTH-based pharmaceutical component havingan excellent stability. TABLE 4 Stability of hPTH(1-34) (storage at 80°C. for 15 hours) Decomposition Acetic acid content (%) products 2.0 2.53.6 5.1 6.2 7.5 9.1 9.9 12.5 13.7 18.3* 72.5* X1 (%) ND ND ND ND ND NDND ND ND ND ND ND X2 (%) ND ND ND ND ND ND ND ND ND ND ND ND  B (%) 0.150.43 0.64 0.91 0.95 1.32 2.19 1.87 1.82 1.93 2.75 5.24  C (%) 0.09 0.280.61 1.30 1.63 2.85 4.92 4.80 6.25 6.92 17.8 19.5  D (%) 0.16 0.27 0.400.60 0.87 1.04 1.50 1.58 1.28 1.31 1.82 2.8 Others (%) 0.74 0.93 1.101.14 1.48 2.22 1.45 2.54 1.91 2.58 12.4 39.9 hPTH(1-34) 98.9 98.1 97.296.0 95.1 92.6 89.9 89.2 88.7 87.3 65.2 32.6 (%)ND: Not detected*Occurs as a liquid after storage.X1: [Met(O)⁸]-hPTH(1-34)X2: [Met(O)¹⁸]-hPTH(1-34)B: Mixture of [Nε-AcLys¹⁸]-hPTH(1-34) and [Nε-AcLys²⁶]-hPTH (1-34)C: [Nα-AcSer¹]-hPTH(1-34)D: [Nε-AcLys²⁷]-hPTH(1-34)

Experiment 3

Stability of hPTH(1-84)

Stored at 80° C. for 15 hours were hPTH(1-84) preparations as obtainedin Example 6 and Reference Example 5 which contained the respectiveacetic acid contents as specified in Example 6 and Reference Example 5;and each of the preparations was left to stand at room temperature forone minute to allow methylene chloride to vaporize, and then capped.Those hPTH(1-84) preparations contained the respective acetic acidcontents were stored at 80° C. for 15 hours; 1 mL of distilled water wasadded to each of the preparations with a syringe for dissolution; andfor each preparation, its content of decomposition products(by-products) was determined before and after storage. The results areshown in Table 5. The reverse phase HPLC chromatogram of the samplehaving undergone storage is shown in FIG. 5. In addition, the contents(%) of decomposition products (by-products) of each hPTH(1-84)preparation after storage is plotted as a function of its acetic acidcontent in FIG. 6.

As shown in FIG. 5, decomposition products (by-products) designated asR1 to R6 were produced as a result of storage. It was demonstrated fromTable 5 and FIG. 6 that decomposition products other than the onedesignated as R5 increase as a function of acetic acid content.

The post-storage purity of hPTH(1-84) preparations was plotted as afunction of their acetic acid contents in FIG. 7. The purity ofhPTH(1-84) preparation increases with the reduction of its acetic acidcontent, and rises sharply when the acetic acid content falls below thechemical equivalent (about 4.5%). From above results it was suggestedalthough the hPTH(1-84) preparation contains certain decompositionproducts whose content is independent of the content of coexistentacetic acid, for the development of the majority of decompositionproducts is closely involved acetic acid that exists as the constituentof a salt of hPTH(1-84) or as an adherent, as in hPTH(1-34). Namely,reduction of the acetic acid content of an hPTH preparation will lead tothe production of an hPTH-based pharmaceutical component having anexcellent stability. TABLE 5 Stability of hPTH(1-84) (storage at 80° C.for 15 hours) Decomposition Acetic acid content (%) products 1.6 2.2 2.73.8 5.2 6.4 8.0 10.2 12.3 22.9 33.6 R1 (%) ND 2.52 4.38 4.55 5.46 6.606.82 8.04 8.8 12.0 12.4 R2 (%) 0.05 0.24 1.83 1.85 2.67 4.05 3.80 5.035.91 11.3 14.8 R3 (%) ND ND 1.82 1.80 2.00 2.31 2.22 2.28 2.45 3.21 9.32R4 (%) 0.88 1.83 2.18 2.15 2.39 2.57 2.59 2.86 2.96 3.41 6.61 R5 (%)5.68 5.27 4.48 5.11 5.56 4.89 4.62 4.82 5.03 5.89 5.13 R6 (%) 0.68 1.661.66 1.83 2.94 2.83 2.76 3.33 4.70 6.87 12.2 hPTH(1-84) 92.8 88.5 83.682.7 79.0 76.7 77.2 73.6 70.1 57.3 39.6 (%)ND: Not detected

Experiment 4

Sensory Test (1)

For the hPTH-based medicinal component, assessment of its use feelingwas achieved by applying it into the nasal cavity.

The hPTH(1-34) preparation prepared as in Reference Example 1 wasdissolved in 0.6 w/v % aqueous solution of citric acid or in water to 10mg/mL; the solution was transferred in a screw type glass vial; a spraynozzle (50 μL)(Valois Co.) was attached to the viol; 50 μL of thesolution was sprayed into one nasal cavity; and the odor and irritationevoked by nasal spraying were assessed (testers consisting of 12 healthynormal adult males). The odor was ranked in four stages as “acidic odorpresent,” “acidic odor slightly present,” “a slight odor detected,” and“no odor detected” according to the olfactory sensation in the tester.The irritation was ranked in four stages as “painfully irritating,”“strongly irritating,” “weakly irritating,” and “very weaklyirritating.” Then, the odor and irritation were scored according totheir rank. As the control, physiological saline was used. The resultsare shown in Table 6.

As is obvious from Table 6, the hPTH(1-34) preparation dissolved inaqueous solution of citric acid and the aqueous solution of hPTH(1-34)preparation evoked a strong acidic odor to cause a discomfort in thetester.

Then, aqueous solutions of various organic acids were prepared, in orderto identify the factors responsible for the odor and irritation thepreparation evokes when it is applied in the nasal cavity. The organicacid employed included acetic acid, citric acid and tartaric acid;oxalic acid; malic acid; phthalic acid; ascorbic acid; adipic acid; andglycolic acid.

Prepared were 0.1 v/v %, 0.2 v/v %, 0.3 v/v % and 0.6 v/v % aqueoussolutions of acetic acid, and 0.6 w/v % aqueous solutions of citricacid, tartaric acid, oxalic acid, malic acid, phthalic acid, ascorbicacid, adipic acid, and glycolic acid. The solution was sprayed into thenasal cavity; and the odor and irritation were assessed in the samemanner as above (testers consisting of four healthy normal adult males).The results are shown in Table 6. As is obvious from Table 6, 0.3 v/v %or higher concentrated aqueous solutions of acetic acid evoke a strongacidic odor, and the irritating activity therefrom also rapidlyincreases when the acetic acid concentration becomes 0.3 v/v % orhigher.

On the other hand, the aqueous solutions of other organic acids do notevoke any detectable odor, and the irritation evoked in the nasal cavityby the aqueous solution of citric acid, together with those of ascorbicacid, adipic acid and glycolic acid, is the same with that fromphysiological saline.

The hPTH(1-34) preparation as acetic acid content being 9.5% obtained inReference Example 1, and the amount of acetic acid contained in thispreparation was approximately the same with the corresponding values ofthe hPTH(1-34) solution and of 0.1 v/v % aqueous solution of acetic acidused in this test.

From the above results, it is suggested that, since 0.6 w/v % aqueoussolution of citric acid itself does not evoke any detectable odor, theodor evoked by the hPTH preparation dissolved in 0.6 w/v % aqueoussolution of citric acid could be ascribed to acetic acid existing as theconstituent of a salt of hPTH(1-34) or as an adherent to the salt in thepreparation, even though acetic acid as contained in the hPTHpreparation would not evoke any detectable odor, if it exists as anaqueous solution, that is, in combination with water.

The hPTH(1-34) preparation as acetic acid content being 2.9% prepared asin Example 1(3) was dissolved in 0.4 w/v % aqueous solution of citricacid to 5 mg/mL. In the same manner as above, the test solution wasapplied in the nasal cavity, and the odor and irritation evoked therebywere assessed (testers consisting of 12 healthy normal adult males). Theresults are shown in Table 6. As is obvious from Table 6, it isindicated that reducing the content of acetic acid in the preparationwill inhibit the odor and irritability of the preparation, and thus thepreparation which has a reduced content of acetic acid will become apharmaceutical component which will ensure an excellent use feeling whenincorporated into a pharmaceutical composition for practical use. TABLE6 Irritability and odor of aqueous solutions of hPTH(1-34) and ofvarious organic acids hPTH/organic acid Irritability Odor hPTH(1-34)*¹++ Acidic odor present hPTH(1-34)*² + Acidic odor present Physiologicalsaline + No odor present Acetic acid +++ Acidic odor present (0.6 v/v %aqueous solution) Acetic acid +++ Acidic odor present (0.3 v/v % aqueoussolution) Acetic acid + Slight acidic odor present (0.2 v/v % aqueoussolution) Acetic acid + Scarcely any odor present (0.1 v/v % aqueoussolution) Citric acid + No odor present (0.6 w/v % aqueous solution)Tartaric acid ++++ No odor present (0.6 w/v % aqueous solution) Oxalicacid ++++ No odor present (0.6 w/v % aqueous solution) Malic acid ++++No odor present (0.6 w/v % aqueous solution) Phthalic acid +++ No odorpresent (0.6 w/v % aqueous solution) Ascorbic acid + No odor present(0.6 w/v % aqueous solution) Adipic acid + No odor present (0.6 w/v %aqueous solution) Glycolic acid + No odor present (0.6 w/v % aqueoussolution) hPTH(1-34)*³ + Slight acidic odor present++++: Painfully irritation,+++: Strong irritation++: Weak irritation+: Very weak irritation*¹10 mg/mL hPTH(1-34) solution (acetic acid content being 9.5%)dissolved in 0.6 w/v % aqueous solution of citric acid*²10 mg/mL aqueous solution of hPTH(1-34) (acetic acid content being9.5%)*³5 mg/mL hPTH(1-34) solution (acetic acid content being 2.9%) dissolvedin 0.4 w/v % aqueous solution of citric acid

Experiment 5

Sensory Test (2)

(1) Preparation of Test Solutions

An amount of preparation corresponding to 300 mg as hPTH(1-34) obtainedas acetic acid content being about 9.5% in Reference Example 1 wasdissolved in distilled water (30 mL), to give an aqueous solution ofhPTH(1-34) at 10 mg/mL. The solution (pH4.7) was subjected toelectrodialysis for removal of acetic acid using a micro acilyser (AsahiKasei Corp.) incorporating an electrodialysis membrane AC-130-10 (AsahiKasei Corp.) until the dialysis solution had a pH of 5.0 (acetic acidcontent being 7.3%). An aqueous solution of hPTH(1-34) considerablyremoved of acetic acid was obtained.

Some aqueous solution of hPTH(1-34) was similarly subjected toelectrodialysis until pH6.0 was reached (acetic acid content being2.9%), to give an aqueous solution of hPTH(1-34) considerably removed ofacetic acid. A same aqueous solution of hPTH(1-34) was similarly treateduntil pH 7.6 was reached (acetic acid content being 0.5%), to give anaqueous solution of hPTH(1-34) largely removed of acetic acid.

(2) Sensory Test

To 1.5 mL of each test solution prepared in this testing example (1),was added 1.5 mL of aqueous solution containing 270 mg of purifiedsucrose, 12 mg of citric acid, and 0.3 mg of benzalkonium chloride, andthe mixture was employed as a test solution for intranasal application.The solution was transferred in a screw type glass vial with a stopper;and a spray nozzle (50 μL)(Valois) was attached to the vial for the test(testers consisting of 5 healthy normal adult males). The odor wasclassified as “A: scarcely any acetic acid odor detected,” “B: weakacetic acid odor detected,” and “C: strong acetic acid odor detected.”The irritation was classified as “A: no irritation felt,” “B: more orless irritation felt,” and “C: strong irritation felt.” Then, the odorand irritation were scored according to their classification. Theresults are shown in Table 7.

As is obvious from Table 7, it is indicated that reducing the content ofacetic acid in the preparation will inhibit the odor and irritability ofthe preparation, and thus the preparation which has a reduced content ofacetic acid will become a pharmaceutical component which will ensure anexcellent use feeling to be suitable for a protracted use whenincorporated into a pharmaceutical composition for practical use. TABLE7 Sensory test of hPTH(1-34)-based pharmaceutical products containingvarious amounts of acetic acid Acetic acid Acetic acid Acetic acidAcetic acid content content content content 9.5% 7.3% 2.9% 0.5% Irrita-Irrita- Irrita- Irrita- Subject Odor bility Odor bility Odor bility Odorbility {circle over (1)} B B B B B B A A {circle over (2)} B B A B A B AB {circle over (3)} C B B A A A A A {circle over (4)} C B B A A A A A{circle over (5)} A B B A B A A AOdorA: Scarcely any acidic odor detectedB: Weak acidic odor detectedC: Strong acidic odor detectedIrritabilityA: No irritation feltB: More or less irritation feltC: Strong irritation felt

Experiment 6

Effects of Various Organic Acids on the Stability of hPTH Preparations

Removal of acetic acid content from an hPTH preparation may be achievedby electrodialysis as mentioned earlier, but it may be achieved byreplacing the acetic acid with another organic acid.

If the acetic acid component of an hPTH preparation is replaced withanother organic acid, how the newly introduced organic acid will affectthe stability of the hPTH preparation was assessed.

The organic acid used in this test included adipic acid, citric acid andglycolic acid which are known as absorption stimulants, and had beenfound in Testing Example 4 as giving a good use feeling. The organicacid the addition of which was intended to replace acetic acid boundwith hPTH was added at a concentration equal to the chemical equivalentof acetic acid. An hPTH(1-34) molecule contains nine basic amino acidresidues and four acidic amino acid residues, that is, an hPTH(1-34)molecule has five positive charges available for binding with acid toform a salt therewith. Thus, with one mol of hPTH(1-34) (Mw. 4117.8)will bind 5/2 mol of adipic acid (Mw. 146.14), 5/3 mol of citric acid(Mw. 192.13), or 5 mol of gylicolic acid (Mw. 76.05), to form a salt.Adipic acid, citric acid or glycolic acid was added to the test hPTHpreparation, so as to satisfy the respective mol proportion describedabove.

A hPTH(1-34) preparation as acetic acid content being 2.9% obtained inExample 1(3) was used to prepare an aqueous solution of hPTH(1-34) at 5mg/mL.

To 2 mL of this solution (10 mg of hPTH(1-34) or 2.43 μmol), was added888 μg of adipic acid (2.43×5/2 μmol), 778 μg of citric acid (2.43×5/3μmol), or 924 μg of glycolic acid (2.43×5 μmol). Each solution wasadjusted with distilled water such that the resulting solution containedthe peptide at 1 mg/mL. The solution was lyophilized, to give alyophilized sample containing 10 mg of hPTH(1-34). For samples thusprepared, some were subjected to reverse phase HPLC to give apre-storage purity of hPTH; others were stored at 60° C. for threeweeks; then they were similarly subjected to reverse phase HPLC, to givea post-storage purity of hPTH; and the pre- and post-storage purityvalues were compared to assess the stability of the hPTH preparation. AnhPTH(1-34) preparation as acetic acid content being 2.9% prepared inExample 1(3) was used as control. The results are shown in Table 8.

While the post-storage purity of the hPTH preparation as acetic acidcontent being 2.9% was 92.7%, the same preparation, when receiving theaddition of adipic acid, citric acid or glycolic acid before storage,showed post-storage purity values of 92.0, 93.9 and 90.3%, respectively.It is indicated from this that an hPTH pharmaceutical componentsupplemented with adipic acid, citric acid or glycolic acid will behighly stable, and ensure a good use feeling like an hPTH pharmaceuticalcomponent in which the content of acetic acid existing as theconstituent of a salt of hPTH or an adherent has been reduced.

It is also indicated that a pharmaceutical component can be obtained byreplacing acetic acid existing in an hPTH preparation as the constituentof a salt or an adherent with a certain organic acid, will serve as apharmaceutical component similarly to an hPTH preparation whose aceticacid content has been reduced. It is further indicated that thecomponent, which is highly stable and will ensure an excellent usefeeling if incorporated into a pharmaceutical composition, has also afeature of compatibly receiving the addition of an organic acid whichmay be added to improve the absorption of the component. TABLE 8 Effectsof various organic acids on the stability of hPTH (storage at 60° C./3weeks) Glycolic Acetic acid Adipic acid Citric acid acid hPTH(1-34) 9.5%2.9% 8.8% 7.7% 9.2% Acid content* Purity before 99.8% 99.8% 100.0%100.0% 100.0% storage (%) Purity after 73.2% 92.7% 92.0% 93.9% 90.3%storage (%)*Acid content = acid weight × 100(%)/peptide weight of hPTH

Experiment 7

Test of Absorption Through Nasal Mucosa

When designing a pharmaceutical composition for intranasaladministration from the preparation, the absorption of the preparationthrough the nasal mucosa becomes an important factor. In view of this,the absorption of the preparation through the nasal mucosa was tested.The effects of citric acid and ascorbic acid which were found in TestingExample 4 to give a good use feeling when added to the hPTH preparation,on the nasal absorption of hPTH were assessed by following the areasunder curve (AUC) of an hPTH plasma concentration-time curve, and thebioavailability of hPTH.

hPTH(1-34) preparation as acetic acid content being 2.9% prepared inExample 1(3) were dissolved in 0.3 and 0.6 w/v % aqueous solutions ofascorbic acid, and in 0.2, 0.3, 0.4 and 0.6 w/v % aqueous solutions ofcitric acid, to give hPTH solutions at 5 mg/mL. Similarly, an hPTH(1-34)preparation as acetic acid content being 0.9% prepared in Example 5(4)was dissolved in 0.6 w/v % aqueous solution of citric acid, to give apeptide solution at 10 mg/mL. As control were used an hPTH(1-34)preparation as acetic acid content being 2.9% prepared in Example 1(3)and an hPTH(1-34) preparation as acetic acid content being 0.9% preparedin Example 5(4), both being dissolved in physiological saline.

Further, hPTH(1-34) preparations as acetic acid content being 2.9%prepared in Example 1(3) were dissolved in 0.3 w/v % or 0.6 w/v %aqueous solution of citric acid; to this solution was added camostatmesilate known as an inhibitor of proteinase to 0.3 w/v %; and theresulting solution was used for the test.

Seven to nine week old Sprague-Dawley male rats (Crj:CD, Charles RiverJapan, Inc.) were kept in metal cages at 22±5° C. and 30 - 70% relativehumidity with a dark-light cycle changing at 12 hour intervals, beingallowed to freely fed on food pellets and tap water. For twenty-fourhours prior to test, they had been fasted (a group consisting of fiverats).

For intranasal administration, the rat, while being kept underpentobarbital anesthesia, had a cannule inserted through a femoralartery; and 5 μL of test solution, or 10 μL of benzalkonium chloridecontaining sucrose was administered into the nasal cavity with aPipetman (TM). Blood was sampled through the cannule into a tubecontaining an anticoagulant and proteinase inhibitor; and the blood wascentrifuged to give plasma. The concentrations of hPTH(1-34) andhPTH(1-84) in plasma were determined by RIA using anti PTH(1-34)antibodies (Chemicon International Inc.)

For subcutaneous administration, the rat received on its back thesubcutaneous injection of the test solution at 1 mL/kg, and theconcentration of hPTH in plasma was determined in the same manner as innasal administration.

The bioavailabilities of hPTH(1-34) and hPTH(1-84) were obtained bycalculation from the ratios of the plasma concentrations three(hPTH(1-34)) or six (hPTH(1-84)) hours after subcutaneous administrationrespectively against the corresponding AUCs of a plasmaconcentration-time curve. The results are shown in Table 9.

Although the bioavailability of hPTH(1-34) subcutaneously applied was1.4% when hPTH(1-34) was used alone, it increased to 5 to 10%, or 12 to19% when it was used as a solute of 0.3 to 0.6 w/v % ascorbic acid, orof 0.2 to 0.6 w/v % citric acid.

The bioavailability of hPTH(1-84) was about 30% when it was used as asolute of 0.6 w/v % aqueous solution of citric acid.

Further, when hPTH(1-34) was applied being dissolved in a solutionsupplemented with camostat mesilate or a proteinase inhibitor, itsbioavailability was 27 to 31%.

From this it was indicated that addition of the organic acids to a smallconcentration notably improves the absorption of hPTH preparationsthrough the nasal mucosa. It was also recognized addition of theabsorption stimulator further improves the nasal absorption of hPTHpreparations. In conclusion, it was demonstrated an hPTH-basedpharmaceutical composition incorporating an hPTH-based pharmaceuticalcomponent intended for intranasal use is suitably used as such becausethe pharmaceutical component is highly stable, and ensures an excellentuse feeling when administered intra-nasally, as the acetic acid contentthereof existing as the constituent of a salt or as an adherent beingdeliberately reduced. TABLE 9 Nasal absorption of hPTH(1-34) andhPTH(1-84) (rat) AUC Bioavailability Prescription Dose Route (pg ×hr/mL) (% s.c.) hPTH(1-34)  25 μg/kg Subcutaneous  1356.5 ± 503.4 (100)(Saline) hPTH(1-34) 125 μg/kg Intra-nasal   93.7 ± 61.3 1.4 (Saline) 0.3w/v % Ascorbic 125 μg/kg Intra-nasal  357.4 ± 308.3 5.2 acid 0.6 w/v %Ascorbic 125 μg/kg Intra-nasal  687.3 ± 281.9 9.9 acid 0.2 w/v % Citricacid 125 μg/kg Intra-nasal  838.4 ± 407.9 12.2 0.3 w/v % Citric acid 125μg/kg Intra-nasal  1208.9 ± 618.6 17.6 0.4 w/v % Citric acid 125 μg/kgIntra-nasal  1285.2 ± 572.4 18.7 0.6 w/v % Citric acid 125 μg/kgIntra-nasal  1254.9 ± 526.1 18.3 0.3 w/v % Citric acid 125 μg/kgIntra-nasal  2118.0 ± 860.4 30.8 + 0.3 w/v % Camostat 0.6 w/v % Citricacid 125 μg/kg Intra-nasal  1829.4 ± 1072.7 27.0 + 0.3 w/v % CamostathPTH(1-84) 285 μg/kg Subcutaneous 46833.3 ± 15667 (100) (Saline)hPTH(1-84) 285 μg/kg Intra-nasal 14166.7 ± 6833 30.2 0.6 w/v % Citricacid

Formulation Example 1

40.5 g of purified sucrose (Japanese Pharmacopoeia) was dissolved in124.2 g of purified water (Japanese Pharmacopoeia) to prepare 150 mL ofa 27 w/v % aqueous solution of purified sucrose. On the other hand, anamount of preparation corresponding to 1.5 g as hPTH(1-34) obtained asacetic acid content being 2.1% (ten vials) which was obtained in Example2, was dissolved in about 75 mL of purified water (JapanesePharmacopoeia) as a pharmaceutical component. A concentration of hPTH(1-34) in the solution was determined as 20.4 mg/mL by reverse phaseHPLC. 25.9 mL of purified water was added to 72.0 mL of the solution toadjust the concentration to 15 mg/mL. 97 mL of 15 mg/mL hPTH (1-34)solution thus prepared, was taken and was mixed with 48.5 mL of the 27w/v % purified sucrose aqueous solution which was prepared previously toobtain about 145 mL of hPTH (1-34) aqueous solution having a purifiedsucrose concentration of 9 w/v % and a hPTH (1-34) concentration of 10mg/mL. 3mL of the solution was charged into each of 47 vials, and wasfreeze-dried in a freeze-drier, Model FZ-6 (Labconco Corporation), toobtain a stable pharmaceutical composition containing 270mg of purifiedsucrose and 30 mg of hPTH (1-34), per vial.

Formulation Example 2

40.5 g of purified sucrose (Japanese Pharmacopoeia) was dissolved in124.2 g of purified water (Japanese Pharmacopoeia) to prepare 150 mL ofa 27 w/v % aqueous solution of purified sucrose. On the other hand, aspharmaceutical component, an amount of preparation corresponding to 750mg as hPTH(1-34) obtained as acetic acid content being 2.1% (five vials)which was obtained in Example 2, was dissolved in 146 mL of purifiedwater (Japanese Pharmacopoeia) to obtain an aqueous solution of hPTH(1-34) having a hPTH (1-34) concentration of 5.1 mg/mL. Further, 54 mLof purified water (Japanese Pharmacopoeia) was added to the aqueoussolution and agitated thoroughly to adjust the hPTH (1-34) concentrationto 3.8 mg/mL (200 mL). 200 mL of the obtained solution with a hPTH(1-34) concentration of 3.8mg/mL was mixed with 100 mL of the 27 w/v %purified sucrose aqueous solution which was prepared previously, toobtain about 300 mL of a hPTH (1-34) aqueous solution having a purifiedsucrose concentration of 9 w/v % and a hPTH (1-34) concentration of2.5mg/mL. 3mL of the solution was charged into each of 90 vials, and wasfreeze-dried in a freeze-drier, Model FZ-6 (Labconco Corporation), toobtain a stable pharmaceutical composition containing 270 mg of purifiedsucrose and 7.5 mg of hPTH (1-34), per vial.

Formulation Example 3

22.5 g of mannitol (Japanese Pharmacopoeia) was dissolved in 124.2 g ofpurified water (Japanese Pharmacopoeia) to prepare 150 mL of a 15%aqueous solution of mannitol. On the other hand, an amount ofpreparation corresponding to 1.5 g as hPTH(1-34) obtained as acetic acidcontent being 2.1% (ten vials), which was obtained in Example 2, wasdissolved in about 75 mL of purified water (Japanese Pharmacopoeia) as apharmaceutical component. A concentration of hPTH (1-34) in the solutionwas determined as 20.4 mg/mL by reverse phase HPLC. 25.9 mL of purifiedwater was added to 72.0 mL of the solution to adjust the concentrationto 15 mg/mL. 97 mL of 15 mg/mL hPTH (1-34) solution thus prepared, wastaken and was mixed with 48.5 mL of the 15% mannitol aqueous solutionwhich was prepared previously to obtain about 145 mL of hPTH (1-34)aqueous solution having a mannitol concentration of 5% and a hPTH (1-34)concentration of 10 mg/mL. 3mL of the solution was charged into each of47 vials, and was freeze-dried in a freeze-drier, Model FZ-6 (LabconcoCorporation), to obtain a stable pharmaceutical composition containing150 mg of mannitol and 30 mg of hPTH (1-34), per vial.

Formulation Example 4

As a pharmaceutical component, 805 mg (powder weight) of a lyophilizedproduct of hPTH (1-34) preparation as acetic acid content being 2.1%,which was obtained in Example 2, was weighed out and was dissolved in360 mL of purified water (Japanese Pharmacopoeia) to obtain an aqueoussolution with a hPTH (1-34) concentration of 2 mg/mL as determined byreverse phase HPLC. On the other hand, lg of mannitol (JapanesePharmacopoeia) was weighed out and was dissolved in 50 mL of purifiedwater (Japanese Pharmacopoeia) to prepare a mannitol aqueous solution.

The whole of 50 mL of the mannitol aqueous solution and 50 mL of thehPTH (1-34) aqueous solution (2 mg/mL), which was prepared previously,were mixed well. 1 mL of the solution was dispensed into each of vialsand was freeze-dried in a freeze-drier, Model FZ-6 (LabconcoCorporation), to obtain a stable medicinal composition containing 1 mgof hPTH (1-34) and 10 mg of mannitol, per vial.

Formulation Example 5

As a pharmaceutical component, 805 mg (powder weight) of a lyophilizedproduct of hPTH (1-34) preparation as acetic acid content being 2.1%,which was obtained in Example 2, was weighed out and was dissolved in360 mL of purified water (Japanese Pharmacopoeia) to obtain an aqueoussolution with a hPTH (1-34) concentration of 2 mg/mL as determined byreverse phase HPLC. On the other hand, 5 g of mannitol (JapanesePharmacopoeia) was weighed out and was dissolved in 50 mL of purifiedwater (Japanese Pharmacopoeia) to prepare a mannitol aqueous solution.

The whole of 50 mL of the mannitol aqueous solution and 50 mL of thehPTH (1-34) aqueous solution (2 mg/mL), which was prepared previously,were mixed well. 1 mL of the solution was dispensed into each of vialsand was freeze-dried in a freeze-drier, Model FZ-6 (LabconcoCorporation), to obtain a stable pharmaceutical composition containing 1mg of hPTH (1-34) and 50 mg of mannitol, per vial.

Formulation Example 6

As a pharmaceutical component, about 11 mg (powder weight) of alyophilized product of hPTH (1-34) preparation as acetic acid contentbeing 2.1%, which was obtained in Example 2, was weighed out, and waterfor injection (WFI) (Japanese Pharmacopoeia) was added thereto to adjustthe volume to 500 mL to obtain an hPTH (1-34) aqueous solution of 20μg/mL as determined by reverse phase HPLC (solution A). 5 g of purifiedsucrose (Japanese Pharmacopoeia) and 100 mg of benzethonium chloridewere dissolved in WFI (Japanese Pharmacopoeia) and a volume was adjustedto 100 mL (solution B). 30 mL each of the solutions A and B were mixed.The resulting solution was dispensed into each of vials by 1 mL pervial, and freeze-dried in a freeze-drier, Model FZ-6 (LabconcoCorporation), to obtain a stable pharmaceutical composition containing10 μg of hPTH (1-34), 25 mg of purified sucrose and 0.5 mg ofbenzethonium chloride, per vial.

Formulation Example 7

As a pharmaceutical component, about 50 mg (powder weight) of alyophilized product of hPTH (1-84) preparation as acetic acid contentbeing 2.5%, which was obtained in Example 5(2), was weighted out, and aninjection solvent (Japanese Pharmacopoeia) was added thereto to obtain ahPTH (1-84) aqueous solution of 2 mg/mL as determined by reverse phaseHPLC (25 mL).

On the other hand, WFI (Japanese Pharmacopoeia) was added to 2 g ofpurified sucrose (Japanese Pharmacopoeia) to obtain 100 mL of a purifiedsucrose aqueous solution. 20 mL of the hPTH (1-84) aqueous solution (2mg/mL) and 20 mL of the prepared purified sucrose aqueous solution (2w/v %) were mixed. The resulting solution was charged into each of 35vials by lmL per vial and was freeze-dried in a freeze-drier, Model FZ-6(Labconco Corporation), to obtain a stable pharmaceutical compositioncontaining 1 mg of hPTH (1-84) and 10 mg of purified sucrose, per vial.

Formulation Example 8

An attached solvent, used for the pharmaceutical composition obtained inthe aforementioned formulation examples 1 through 7 in adissolved-upon-use type preparation, was prepared as follows.

0.35 g of benzalkonium chloride (Japanese Pharmacopoeia) and 21.0 g ofcitric acid (Japanese Pharmacopoeia) were weighed out and dissolved in350 mL of purified water. 3 mL of the solution, thus obtained, wasdispensed into each of polypropylene bottles to prepare an attachedsolvent.

Formulation Example 9

An attached solvent, used for the pharmaceutical composition obtained inthe aforementioned formulation examples 1 through 7 in adissolved-upon-use type preparation, was prepared as follows.

0.70 g of benzethonium chloride (Japanese Pharmacopoeia) and 14.0 g ofcitric acid (Japanese Pharmacopoeia) were weighed out and dissolved in3500 mL of purified water. 3 mL of the solution thus obtained wasdispensed into each of polypropylene bottles to prepare an attachedsolvent.

Formulation Example 10

An attached solvent, used for the pharmaceutical composition obtained inthe aforementioned formulation examples 1 through 7 in adissolved-upon-use type preparation, was prepared as follows.

0.35 g of benzethonium chloride (Japanese Pharmacopoeia) and 21.0 g ofadipic acid (Japanese Pharmacopoeia) were weighed out and dissolved in3500 mL of purified water. 3 mL of the solution thus obtained wasdispensed into polypropylene bottles to prepare an attached solvent.

Formulation Example 11

An attached solvent, used for the pharmaceutical composition obtained inthe aforementioned formulation examples 1 through 7 in adissolved-upon-use type preparation, was prepared as follows.

0.70 g of cetylpyridium chloride (Japanese Pharmacopoeia) and 14.0 g ofadipic acid (Japanese Pharmacopoeia) were weighed out and dissolved in3500 mL of purified water. 3 mL of the solution thus obtained wasdispensed into polypropylene bottles to prepare an attached solvent.

Formulation Example 12

As a pharmaceutical component, an amount of preparation corresponding to900 mg as hPTH (1-34) obtained as acidic content being 2.5% in Example4(1) (six vials) was dissolved in 18 mL of an WFI (JapanesePharmacopoeia) (50 mg/mL). On the other hand, 12 g of citric acid(Japanese Pharmacopoeia) was dissolved in an injection solvent (JapanesePharmacopoeia) to obtain 1000 mL of a solution (1.2 w/v % citric acidaqueous solution). Using the resulting solutions, liquid medicamentswere obtained as follows.

1. Preparation of pH3, hPTH (1-34) 5 mg/mL, 0.6 w/v % Citric AcidSolution

6mL of the 1.2 w/v % citric acid aqueous solution and 1.2 mL of the 50mg/mL hPTH (1-34) aqueous solution were mixed and a pH of the resultingsolution was adjusted to 3 by adding 95 μL of iN NaOH. Then, WFI wasadded to the solution to make a solution volume 12 mL. The solution,thus obtained, was filtered through a 0.22 μm filter to obtain the titlepharmaceutical composition.

2. Preparation of pH3.5, hPTH (1-34) 5 mg/mL, 0.6 w/v % Citric AcidSolution

6 mL of the 1.2 w/v % citric acid aqueous solution and 1.2 mL of the 50mg/mL hPTH (1-34) aqueous solution were mixed and a pH of the solutionwas adjusted to 3.5 by adding 210 μL of iN NaOH. Then, an WFI was addedto the solution to make a solution volume 12 mL. The solution, thusobtained, was filtered through a 0.22 μm filter to obtain the titlepharmaceutical composition.

3. Preparation of pH4, hPTH (1-34) 5 mg/mL, 0.6 w/v % Citric AcidSolution

6mL of the 1.2 w/v % citric acid aqueous solution and 1.2 mL of the 50mg/mL hPTH (1-34) aqueous solution were mixed and a pH of the solutionwas adjusted to 4.0 by adding 350 μL of 1N NaOH. Then, WFI was added tothe solution to make a solution volume 12 mL. The solution, thusobtained, was filtered through a 0.22 μm filter to obtain the titlepharmaceutical composition.

4. Preparation of pH4.5, hPTH (1-34) lmg/mL, 0.4 w/v % Citric AcidSolution

4 mL of the 1.2 w/v % citric acid aqueous solution, 0.24 mL of the 50mg/mL hPTH (1-34) aqueous solution, and about 5 mL of purified waterwere mixed and a pH of the solution was adjusted to 4.5 by adding 335 μLof 1N NaOH. Then, WFI was added to the solution to make a solutionvolume 12 mL. The solution, thus obtained, was filtered through a 0.22μm filter to obtain the title pharmaceutical composition.

INDUSTRIAL APPLICABILITY

According to this invention, it is provided a pharmaceutical componentwhich, being reduced of its content of acetic acid, is highly stable andwill ensure an excellent use feeling when incorporated into apharmaceutical component for usage.

The pharmaceutical component of this invention can tolerate the additionof appropriate amounts of various functional components, as well as acarrier or excipient which is usually used during pharmaceuticalpreparation, may be incorporated into pharmaceutical compositions ofwidely varied dosage forms, or may be shaped into widely varied dosageforms itself.

According to this invention, a pharmaceutical composition for intranasaladministration is provided which is usable over a long period.

1. A pharmaceutical component comprising a human parathyroid hormonepeptide or its derivative, and acetic acid contained in an amount lessthan its chemical equivalent with respect to the human parathyroidhormone peptide or to its derivative.
 2. A pharmaceutical component asdescribed in claim 1 comprising a salt of the human parathyroid hormonepeptide or of its derivative with acetic acid wherein acetic acid iscontained in an amount less than its chemical equivalent with respect tothe human parathyroid hormone peptide or to its derivative.
 3. Apharmaceutical component as described in claim 1, wherein the humanparathyroid hormone peptide or its derivative is a peptide comprisingamino acid residues designated as amino acid Nos. 1-84.
 4. Apharmaceutical component as described in claim 10, wherein thepharmaceutical component comprises 4 weight % or less acetic acid withrespect to the weight of the peptide.
 5. A pharmaceutical component asdescribed in claim 1, wherein the pharmaceutical component is alyophilized composition.
 6. A pharmaceutical component as described inclaim 1, wherein the pharmaceutical component is for intranasaladministration.
 7. A pharmaceutical composition for intranasaladministration comprising a human parathyroid hormone peptide or itsderivative and acetic acid contained in an amount less than its chemicalequivalent with respect to the human parathyroid hormone peptide or toits derivative.
 8. A prior-to-use dissolvable pharmaceutical productcomprising a lyophilized portion and a dissolving solution portionattached thereto wherein the pharmaceutical component as described inclaim 1 is contained in the lyophilized portion.
 9. A pharmaceuticalcomponent as described in claim 3, wherein the pharmaceutical componentcomprises 3 weight % or less acetic acid with respect to the weight ofthe peptide.
 10. A pharmaceutical component as described in claim 1,wherein the human parathyroid hormone peptide or its derivative is apeptide comprising amino acid residues designated as amino acid Nos.1-34.
 11. A pharmaceutical component as described in claim 10, whereinthe pharmaceutical component comprises 6 weight % or less acetic acidwith respect to the weight of the peptide.